Targeting microRNAs for the treatment of liver cancer

ABSTRACT

Provided herein are methods for the treatment of liver cancer. These methods encompass the administration of a compound comprising a modified oligonucleotide, wherein the modified oligonucleotide is targeted to a miRNA. Also provided herein are compositions for the treatment of liver cancer. Such compositions include compounds comprising a modified oligonucleotide, wherein the modified oligonucleotide is targeted to a miRNA. Certain miRNAs have been identified as overexpressed in liver cancer, such as, for example, hepatocellular carcinoma, and are thus selected for targeting by modified oligonucleotides. Further, certain miRNAs have been identified as overexpressed in hepatocellular carcinoma cells exposed to dioxin, and are thus selected for targeting by modified oligonucleotides. Antisense inhibition of certain of these miRNAs has been found to inhibit cell proliferation and induce apoptosis.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 15/334,422, filed Oct. 26, 2016, which is a divisional of U.S.patent application Ser. No. 14/833,889, filed Aug. 24, 2015, now U.S.Pat. No. 9,506,062, which is a divisional of U.S. patent applicationSer. No. 14/168,812, filed Jan. 30, 2014, now U.S. Pat. No. 9,150,857,which is a divisional of U.S. patent application Ser. No. 13/481,105,filed May 25, 2012, now U.S. Pat. No. 8,680,067, which is a divisionalof U.S. patent application Ser. No. 12/740,211, filed Apr. 28, 2010, nowU.S. Pat. No. 8,211,867, which is a § 371 national entry ofInternational Application No. PCT/US2008/081645, filed Oct. 29, 2008,which claims priority under 35 U.S.C. § 119(e) to U.S. ProvisionalApplication No. 60/983,231, filed Oct. 29, 2007, each of which isincorporated by reference herein in its entirety for any purpose.

INCORPORATION OF SEQUENCE LISTING

The present application is filed with a Sequence Listing in electronicformat. The Sequence Listing is provided as a file entitled“2015-08-24_01138-0003-03US_Sequence_Listing_ST25.txt” created on Aug.6, 2015, which is 8,147 bytes in size. The information in the electronicformat of the sequence listing is incorporated herein by reference inits entirety.

FIELD OF INVENTION

Provided herein are methods and compositions for the treatment of livercancer, including but not limited to hepatocellular carcinoma. Providedherein are also methods and compositions for the treatment of dioxininduced liver cancer, including but not limited to dioxin inducedhepatocellular carcinoma. Such methods comprise administering a compoundcomprising a modified oligonucleotide targeted to a miRNA.

DESCRIPTION OF RELATED ART

MicroRNAs (miRNAs), also known as “mature miRNA” are small(approximately 18-24 nucleotides in length), non-coding RNA moleculesencoded in the genomes of plants and animals. In certain instances,highly conserved, endogenously expressed miRNAs regulate the expressionof genes by binding to the 3′-untranslated regions (3′-UTR) of specificmRNAs. More than 1000 different miRNAs have been identified in plantsand animals certain mature miRNAs appear to originate from longendogenous primary miRNA transcripts (also known as pri-miRNAs,pri-mirs, pri-miRs or pri-pre-miRNAs) that are often hundreds ofnucleotides in length (Lee, et al., EMBO J., 2002, 21(17), 4663-4670).

Functional analyses of miRNAs have revealed that these small non-codingRNAs contribute to different physiological processes in animals,including developmental timing, organogenesis, differentiation,patterning, embryogenesis, growth control and programmed cell death.Examples of particular processes in which miRNAs participate includestem cell differentiation, neurogenesis, angiogenesis, hematopoiesis,and exocytosis (reviewed by Alvarez-Garcia and Miska, Development, 2005,132, 4653-4662). In some instances, miRNAs are thought to exercisepost-transcriptional control in most eukaryotic organisms and have beendetected in plants and animals as well as certain viruses.

Families of miRNAs can be characterized by nucleotide identity atpositions 2-8 of the miRNA, a region known as the seed sequence. Lewiset al. describe several miRNA families, as well as miRNA superfamilies,which are characterized by related seed sequences (Lewis et al. Cell.2005, 120(1):15-20).

SUMMARY OF INVENTION

In certain embodiments, the present invention provides methods fortreating liver cancer, comprising administering to a subject in needthereof a compound comprising a modified oligonucleotide consisting of15 to 30 linked nucleosides, wherein the modified oligonucleotide has anucleobase sequence that is complementary to a nucleobase sequenceselected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, and 8; or to a sequence atleast 80% identical thereto.

In certain embodiments, the present invention provides methods fortreating liver cancer, comprising administering to the subject in needthereof a compound comprising a modified oligonucleotide consisting of15 to 30 linked nucleosides and having a nucleobase sequence that iscomplementary to a nucleobase sequence selected from SEQ ID NOs: 9, 10,11, 12, 13, 14, 15, and 16; or to a nucleobase sequence at least 80%identical thereto.

In certain embodiments, the present invention provides methods fortreating liver cancer, comprising administering to a subject in needthereof a compound comprising a modified oligonucleotide consisting of15 to 30 linked nucleosides, wherein the modified oligonucleotide has anucleobase sequence comprising at least 15 contiguous nucleobases of anucleobase sequence selected from among the nucleobase sequences recitedin SEQ ID NOs 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and30; or a sequence at least 80% identical thereto.

In certain embodiments, the present invention provides methods fortreating liver cancer, comprising administering to a subject in needthereof a pharmaceutical composition comprising a modifiedoligonucleotide consisting of 15 to 30 linked nucleosides, wherein themodified oligonucleotide has a nucleobase sequence that is complementaryto a nucleobase sequence selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7,and 8; or to a sequence at least 80% identical thereto.

In certain embodiments, the present invention provides methods fortreating liver cancer, comprising administering to the subject in needthereof a pharmaceutical composition comprising a modifiedoligonucleotide consisting of 15 to 30 linked nucleosides and having anucleobase sequence that is complementary to a nucleobase sequenceselected from SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, and 16; or to anucleobase sequence at least 80% identical thereto.

In certain embodiments, the present invention provides methods fortreating liver cancer, comprising administering to a subject in needthereof a pharmaceutical composition comprising a modifiedoligonucleotide consisting of 15 to 30 linked nucleosides, wherein themodified oligonucleotide has a nucleobase sequence comprising at least15 contiguous nucleobases of a nucleobase sequence selected from amongthe nucleobase sequences recited in SEQ ID NOs 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, and 30; or a sequence at least 80% identicalthereto.

In certain embodiments, the present invention provides a compoundcomprising a modified oligonucleotide consisting of 15 to 30 linkednucleosides, wherein the modified oligonucleotide has a nucleobasesequence that is complementary to a nucleobase sequence selected fromSEQ ID NO: 1, 2, 3, 4, 5, 6, 7, and 8; or to a sequence at least 80%identical thereto, for use in treating liver cancer.

In certain embodiments, the present invention provides a compoundcomprising a modified oligonucleotide consisting of 15 to 30 linkednucleosides, wherein the modified oligonucleotide has a nucleobasesequence that is complementary to a nucleobase sequence selected fromSEQ ID NO: 9, 10, 11, 12, 13, 14, 15, and 16; or to a sequence at least80% identical thereto, for use in treating liver cancer.

In certain embodiments, the present invention provides a compoundcomprising a modified oligonucleotide consisting of 15 to 30 linkednucleosides, wherein the modified oligonucleotide has a nucleobasesequence comprising at least 15 contiguous nucleobases of a nucleobasesequence selected from among the nucleobase sequences recited in SEQ IDNOs 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30; or to asequence at least 80% identical thereto, for use in treating livercancer.

In certain embodiments, the invention provides methods for treating adioxin induced liver cancer comprising administering to a subject inneed thereof a compound comprising a modified oligonucleotide consistingof 15 to 30 linked nucleosides, wherein the modified oligonucleotide hasa nucleobase sequence that is complementary to a sequence selected fromSEQ ID NO: 31, 32, 33, 34, 35, 36, and 37; or to a sequence at leastabout 80% identical thereto.

In certain embodiments, the invention provides a compound comprising amodified oligonucleotide consisting of 15 to 30 linked nucleosides,wherein the modified oligonucleotide has a nucleobase sequence that iscomplementary to a sequence selected from SEQ ID NO: 31, 32, 33, 34, 35,36, and 37; or to a sequence at least about 80% identical thereto, foruse in treating a dioxin induced liver cancer.

In certain embodiments, the invention provides methods for treating adioxin induced liver cancer comprising administering to a subject inneed thereof a compound comprising a modified oligonucleotide consistingof 15 to 30 linked nucleosides, wherein the modified oligonucleotide hasa nucleobase sequence comprising at least 15 contiguous nucleobases of anucleobase sequence selected from among the nucleobase sequences recitedin SEQ ID NOs 38, 39, and 40; or to a sequence at least about 80%identical thereto.

In certain embodiments, the invention provides a compound comprising amodified oligonucleotide consisting of 15 to 30 linked nucleosides,wherein the modified oligonucleotide has a nucleobase sequencecomprising at least 15 contiguous nucleobases of a nucleobase sequenceselected from among the nucleobase sequences recited in SEQ ID NOs 38,39, and 40; or to a sequence at least about 80% identical thereto, foruse in treating a dioxin induced liver cancer.

In certain embodiments, the dioxin induced liver cancer ishepatocellular carcinoma.

In certain embodiments, the present invention provides a compoundcomprising a modified oligonucleotide consisting of 15 to 30 linkednucleosides and having a nucleobase sequence that is complementary to anucleobase sequence selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, and 8;or to a sequence at least about 80% identical thereto.

In certain embodiments, the present invention provides a compoundcomprising a modified oligonucleotide consisting of 15 to 30 linkednucleosides and having a nucleobase sequence that is complementary to anucleobase sequence selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, and 8;or to a sequence at least about 80% identical thereto, for use intreating liver cancer.

In certain embodiments, the present invention provides a compoundcomprising a modified oligonucleotide consisting of 15 to 30 linkednucleosides and having a nucleobase sequence that is complementary to anucleobase sequence selected from SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15,and 16; or to a sequence at least about 80% identical thereto.

In certain embodiments, the present invention provides a compoundcomprising a modified oligonucleotide consisting of 15 to 30 linkednucleosides and having a nucleobase sequence that is complementary to anucleobase sequence selected from SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15,and 16; or to a sequence at least about 80% identical thereto, for usein treating liver cancer.

In certain embodiments, the present invention provides a compoundcomprising a modified oligonucleotide consisting of 15 to 30 linkednucleosides and having a nucleobase sequence comprising at least 15contiguous nucleobases of a nucleobase sequence selected from among thenucleobase sequences recited in SEQ ID NOs 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, and 30.

In certain embodiments, the present invention provides a compoundcomprising a modified oligonucleotide consisting of 15 to 30 linkednucleosides and having a nucleobase sequence comprising at least 15contiguous nucleobases of a nucleobase sequence selected from among thenucleobase sequences recited in SEQ ID NOs 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, and 30, for use in treating liver cancer.

In certain embodiments, the present invention provides a compoundcomprising a modified oligonucleotide consisting of 15 to 30 linkednucleosides and having a nucleobase sequence comprising at least 15contiguous nucleobases of a nucleobase sequence selected from among thenucleobase sequences recited in SEQ ID NOs 38, 39, and 40, for use intreating liver cancer.

In certain embodiments, the present invention provides a pharmaceuticalcomposition comprising a modified oligonucleotide of the invention or asalt thereof and a pharmaceutically acceptable carrier or diluent.

In certain embodiments, the compound consists of a modifiedoligonucleotide.

In certain embodiments, the modified oligonucleotide consists of 15, 16,17, 18, 19, 20, 21, 22, 23, 24, or 25 linked nucleosides.

In certain embodiments, the nucleobase sequence of the modifiedoligonucleotide has no more than two mismatches to a nucleobase sequenceselected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, and 8. In certainembodiments, the nucleobase sequence of the modified oligonucleotide hasno more than one mismatch to a nucleobase sequence selected from SEQ IDNO: 1, 2, 3, 4, 5, 6, 7, and 8. In certain embodiments, the nucleobasesequence of the modified oligonucleotide has one mismatch to anucleobase sequence selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, and 8.In certain embodiments, the nucleobase sequence of the modifiedoligonucleotide has no mismatches to a nucleobase sequence selected fromSEQ ID NO: 1, 2, 3, 4, 5, 6, 7, and 8.

In certain embodiments, the nucleobase sequence of the modifiedoligonucleotide has no more than two mismatches to a nucleobase sequenceselected from SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, and 16. In certainembodiments, the nucleobase sequence of the modified oligonucleotide hasno more than one mismatch to a nucleobase sequence selected from SEQ IDNOs: 9, 10, 11, 12, 13, 14, 15, and 16. In certain embodiments, thenucleobase sequence of the modified oligonucleotide has one mismatch toa nucleobase sequence selected from SEQ ID NOs: 9, 10, 11, 12, 13, 14,15, and 16. In certain embodiments, the nucleobase sequence of themodified oligonucleotide has no mismatches to a nucleobase sequenceselected from SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, and 16.

In certain embodiments, the nucleobase sequence of the modifiedoligonucleotide has no more than two mismatches to a nucleobase sequenceselected from SEQ ID NOs: 31, 32, 33, 34, 35, 36, and 37. In certainembodiments, the nucleobase sequence of the modified oligonucleotide hasno more than one mismatch to a nucleobase sequence selected from SEQ IDNOs: 31, 32, 33, 34, 35, 36, and 37. In certain embodiments, thenucleobase sequence of the modified oligonucleotide has one mismatch toa nucleobase sequence selected from SEQ ID NOs: 31, 32, 33, 34, 35, 36,and 37. In certain embodiments, the nucleobase sequence of the modifiedoligonucleotide has no mismatches to a nucleobase sequence selected fromSEQ ID NOs: 31, 32, 33, 34, 35, 36, and 37.

In certain embodiments, the modified oligonucleotide has a nucleobasesequence comprising at least 16 contiguous nucleobases of a nucleobasesequence selected from among the nucleobase sequences recited in SEQ IDNOs 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.

In certain embodiments, the modified oligonucleotide has a nucleobasesequence comprising at least 17 contiguous nucleobases of a nucleobasesequence selected from among the nucleobase sequences recited in SEQ IDNOs 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.

In certain embodiments, the modified oligonucleotide has a nucleobasesequence comprising at least 18 contiguous nucleobases of a nucleobasesequence selected from among the nucleobase sequences recited in SEQ IDNOs 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.

In certain embodiments, the modified oligonucleotide has a nucleobasesequence comprising at least 19 contiguous nucleobases of a nucleobasesequence selected from among the nucleobase sequences recited in SEQ IDNOs 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.

In certain embodiments, the modified oligonucleotide has a nucleobasesequence comprising at least 20 contiguous nucleobases of a nucleobasesequence selected from among the nucleobase sequences recited in SEQ IDNOs 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.

In certain embodiments, the modified oligonucleotide has a nucleobasesequence comprising at least 21 contiguous nucleobases of a nucleobasesequence selected from among the nucleobase sequences recited in SEQ IDNOs 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.

In certain embodiments, the modified oligonucleotide has a nucleobasesequence comprising at least 22 contiguous nucleobases of a nucleobasesequence selected from among the nucleobase sequences recited in SEQ IDNOs 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.

In certain embodiments, the modified oligonucleotide has a nucleobasesequence comprising at least 23 contiguous nucleobases of a nucleobasesequence selected from among the nucleobase sequences recited in SEQ IDNOs 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.

In certain embodiments, the modified oligonucleotide has a nucleobasesequence consisting of a nucleobase sequence selected from among thenucleobase sequences recited in SEQ ID NOs 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, and 30.

In certain embodiments, the modified oligonucleotide has a nucleobasesequence comprising at least 16, at least 17, at least 18, at least 19,at least 20, at least 21, at least 22, or at least 23 contiguousnucleobases of a nucleobase sequence from among the nucleobase sequencesrecited in SEQ ID Nos 38, 39, and 40.

In certain embodiments, the modified oligonucleotide comprises one ormore modified sugars, internucleoside linkages, or nucleobases. Incertain embodiments, at least one internucleoside linkage is a modifiedinternucleoside linkage. For example, at least one internucleosidelinkage may be a phosphorothioate internucleoside linkage. In certainembodiments, each internucleoside linkage is a modified internucleosidelinkage. For example, each internucleoside linkage may be aphosphorothioate internucleoside linkage.

In certain embodiments, at least one nucleoside of the modifiedoligonucleotide comprises a modified sugar. In certain embodiments, eachof a plurality of nucleosides comprises a modified sugar. In certainembodiments, each nucleoside of the modified oligonucleotide comprises amodified sugar. In each of these embodiments, the modified sugar may bea 2′-O-methoxyethyl sugar, a 2′-fluoro sugar, a 2′-O-methyl sugar, or abicyclic sugar moiety. In certain embodiments, each of a plurality ofnucleosides comprises a 2′-O-methoxyethyl sugar and each of a pluralityof nucleosides comprises a 2′-fluoro sugar.

In certain embodiments, the modified oligonucleotide comprises at leastone modified nucleobase. In certain such embodiments, the modifiednucleobase is a 5-methylcytosine. In certain embodiments, at least onenucleoside comprises a cytosine, wherein the cytosine is a5-methylcytosine. In certain such embodiments, each cytosine is a5-methylcytosine.

In certain embodiments, the liver cancer is hepatocellular carcinoma. Incertain embodiments, the subject is a human. In certain embodiments, thehepatocellular carcinoma is dioxin-induced.

In certain embodiments, administration of a compound of the inventioncomprises intravenous administration, subcutaneous administration,intratumoral administration, or chemoembolization.

In certain embodiments, the methods of the present invention furthercomprise administering at least one additional therapy. The additionaltherapy may be a chemotherapeutic agent. The chemotherapeutic agent maybe selected from 5-fluorouracil, gemcitabine, doxorubicine, mitomycin c,sorafenib, etoposide, carboplatin, epirubicin, irinotecan andoxaliplatin. The additional therapy may be administered at the sametime, less frequently, or more frequently than a compound orpharmaceutical composition of the invention.

In certain embodiments, the modified oligonucleotide is administered ata dose selected from 50, 75, 100, 125, 150, 175, 200, 225, 250, 275,300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625,650, 675, 700, 725, 750, 775, and 800 mg. The modified oligonucleotidemay be administered one per day, once per week, once per two weeks, onceper three weeks, or once per four weeks.

In certain embodiments, the administration of a compound of theinvention results in reduction of liver tumor size and/or liver tumornumber. In certain embodiments, the administration of a compound of theinvention prevents an increase in tumor size and/or tumor number. Incertain embodiments, the administration of a compound of the inventionprevents, slows, and/or stops metastatic progression. In certainembodiments, the administration of a compound of the invention extendsthe overall survival time of the subject. In certain embodiments, theadministration of a compound of the invention extends theprogression-free survival of the subject. In certain embodiments,administration of a compound of the invention prevents the recurrence ofliver tumors. In certain embodiments, administration of a compound ofthe invention prevents recurrence of liver tumor metastasis. In certainembodiments, administration of a compound of the invention preventsrecurrence of HCC-derived tumors. In certain embodiments, administrationof a compound of the invention prevents recurrence of HCC-derived tumormetastasis.

In certain embodiments, a subject selected for treatment for livercancer has liver lesions. In certain embodiments, a subject selected fortreatment for liver cancer has elevated serum alpha-fetoprotein and/orelevated serum des-gamma-carboxyprothrombin. In certain embodiments, asubject selected for treatment of liver cancer has abnormal liverfunction.

In certain embodiments, administration of a compound of the inventionreduces serum alpha-fetoprotein and/or serumdes-gamma-carboxyprothrombin in a subject having liver cancer. Incertain embodiments, levels of serum alpha-fetoprotein and/or serumdes-gamma-carboxyprothrombin are measured to assess therapeuticefficacy. In certain embodiments, administration of a compound of theinvention improves liver function of the subject.

These and other embodiments of the present invention will becomeapparent in conjunction with the figures, description and claims thatfollow.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1. Differential expression analysis of miRNAs in liver tumorsamples compared to normal liver tissue samples. Data points havingsignificant p-values are enclosed by red circles. Certain miRNA targetsthat were later selected for further study are represented by filledyellow circles. These miRNA targets include miR-21, miR-125a-5p (labeledas miR-125a), miR-191, miR-210, miR-222, miR-378 (labeled as miR-422b),miR-423-3p, and miR-638.

FIGS. 2A and 2B. Inhibition of cell proliferation in liver cancer celllines following treatment with modified oligonucleotides targeted tomiRNAs. Proliferation of both SNU423 (FIG. 2A) and Hep3B (FIG. 2B) celllines was tested after transfection with modified oligonucleotides. Aproliferation assay was performed 72 hours after transfection.Proliferation was measured and compared to proliferation of cellstreated with a negative control oligonucleotide and to proliferation ofuntransfected cells. Modified oligonucleotides complementary to miR-21,miR-125a, miR-191, miR-210, miR-222, miR-378 (labeled as miR-422b),miR-423, and miR-638 resulted in antiproliferative activity.

FIG. 3. Induction of apoptosis in liver cancer cells following treatmentwith modified oligonucleotides targeted to miRNAs. Hep3B cells weretransfected with modified oligonucleotides. The induction of apoptosiswas measured 24 hours after transfection, by testing Caspase 3/7activation. Treatment of cells with modified oligonucleotidescomplementary to miR-21, miR-125a, miR-191, miR-210, miR-378 (labeled asmiR-422b), miR-423, and miR-638 resulted in significant elevation ofCaspase 3/7 activity, indicating an induction of apoptosis.

FIGS. 4A and 4B. Reduction of subcutaneous tumor volume in mice treatedwith modified oligonucleotides. Subcutaneous tumors were induced by theinjection of HepG2 cells into nude mice. Treatment with MOE-modifiedoligonucleotides complementary to miR-21 (FIG. 4A) and miR-210 (FIG. 4B)was shown to reduce tumor volume, relative to saline control treatments.

FIG. 5. Median expression value (in log(fluorescence)) of miRNA in HepG2cells, wherein the X-axis represents cells 48 hours after TCDDtreatment, and the Y-axis represents untreated cells. The dottedparallel lines describe a fold change of two in either direction. Themiddle line describes an identical median expression in both groups ofcells.

MiRNAs with relatively high expression values in the treated cellsinclude hsa-miR-191, hsa-miR-181a, hsa-miR-181b, and hsa-miR-181a*.

FIG. 6: The results of the Dual-Luciferase Reporter Assay are presentedin a bar-chart, in which the Y-axis represents the R/F % ratio ofcontrol vector. Each bar depicts the normalized luminescence as follows:Bar a—p191 (plasmid baring reverse complement seq of miR-191 at the3′UTR of renilla luciferase only with no modified oligonucleotides), Barb—p191 ASO-miR-NC; Bar c—pControl (control plasmid) ASO-miR-191; Bard—p191 ASO-miR-191; Bar e—pControl (no modified oligonucleotides).

FIG. 7. ChIP (Chromatin Immuno Precipitation) assay using a specificantibody for AhR. The Y-axis depicts binding events per 1,000 cells,with the bars with diagonal stripes representing cells treated withTCDD, and the bars with dots representing cells which were not treated.The pair of bars on the left represents the negative control. The twopairs of bars on the right represent the two binding sites for theAhR/Arnt TF on CYP1A1.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in thearts to which the invention belongs. Unless specific definitions areprovided, the nomenclature utilized in connection with, and theprocedures and techniques of, analytical chemistry, synthetic organicchemistry, and medicinal and pharmaceutical chemistry described hereinare those well known and commonly used in the art. In the event thatthere is a plurality of definitions for terms herein, those in thissection prevail. Standard techniques may be used for chemical synthesis,chemical analysis, pharmaceutical preparation, formulation and delivery,and treatment of subjects. Certain such techniques and procedures may befound for example in “Carbohydrate Modifications in Antisense Research”Edited by Sangvi and Cook, American Chemical Society, Washington D.C.,1994; and “Remington's Pharmaceutical Sciences,” Mack Publishing Co.,Easton, Pa., 18th edition, 1990; and which is hereby incorporated byreference for any purpose. Where permitted, all patents, patentapplications, published applications and publications, GENBANKsequences, websites and other published materials referred to throughoutthe entire disclosure herein, unless noted otherwise, are incorporatedby reference in their entirety. Where reference is made to a URL orother such identifier or address, it is understood that such identifierscan change and particular information on the internet can command go,but equivalent information can be found by searching the internet.Reference thereto evidences the availability and public dissemination ofsuch information.

Before the present compositions and methods are disclosed and described,it is to be understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting. It must be noted that, as used in the specification and theappended claims, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise.

Definitions

“Liver cancer” means malignancy of the liver, either a primary cancer ormetastasized cancer. In certain embodiments, liver cancer includes, butis not limited to, cancer arising from hepatocytes, such as, forexample, hepatomas and hepatocellular carcinomas; fibrolamellarcarcinoma; and cholangiocarcinomas (or bile duct cancer).

“Hepatocellular carcinoma” means primary cancer of the liver arisingfrom hepatocytes.

“Dioxin induced liver cancer” means a liver cancer that is caused bydioxin exposure. In certain embodiments, a dioxin-induced liver canceris dioxin-induced hepatocellular carcinoma.

“Subject” means a human or non-human animal selected for treatment ortherapy.

“Subject in need thereof” means a subject identified as in need of atherapy or treatment. In certain embodiments, a subject has livercancer. In such embodiments, a subject has one or more clinicalindications of liver cancer or is at risk for developing liver cancer.

“Administering” means providing a pharmaceutical agent or composition toa subject, and includes, but is not limited to, administering by amedical professional and self-administering.

“Parenteral administration,” means administration through injection orinfusion. Parenteral administration includes, but is not limited to,subcutaneous administration, intravenous administration, orintramuscular administration.

“Subcutaneous administration” means administration just below the skin.

“Intravenous administration” means administration into a vein.

“Intratumoral administration” means administration within a tumor.

“Chemoembolization” means a procedure in which the blood supply to atumor is blocked surgically, mechanically, or chemically andchemotherapeutic agents are administered directly into the tumor.

“Duration” means the period of time during which an activity or eventcontinues. In certain embodiments, the duration of treatment is theperiod of time during which doses of a pharmaceutical agent orpharmaceutical composition are administered.

“Therapy” means a disease treatment method. In certain embodiments,therapy includes, but is not limited to, chemotherapy, surgicalresection, liver transplant, and/or chemoembolization.

“Treatment” means the application of one or more specific proceduresused for the cure or amelioration of a disease. In certain embodiments,the specific procedure is the administration of one or morepharmaceutical agents.

“Amelioration” means a lessening of severity of at least one indicatorof a condition or disease. In certain embodiments, amelioration includesa delay or slowing in the progression of one or more indicators of acondition or disease. The severity of indicators may be determined bysubjective or objective measures which are known to those skilled in theart.

“Prevention” refers to delaying or forestalling the onset or developmentor progression of a condition or disease for a period of time, includingweeks, months, or years.

“Therapeutic agent” means a pharmaceutical agent used for the cure,amelioration or prevention of a disease.

“Chemotherapeutic agent” means a pharmaceutical agent used to treatcancer.

“Chemotherapy” means treatment of a subject with one or morepharmaceutical agents that kills cancer cells and/or slows the growth ofcancer cells.

“Dose” means a specified quantity of a pharmaceutical agent provided ina single administration. In certain embodiments, a dose may beadministered in two or more boluses, tablets, or injections. Forexample, in certain embodiments, where subcutaneous administration isdesired, the desired dose requires a volume not easily accommodated by asingle injection. In such embodiments, two or more injections may beused to achieve the desired dose. In certain embodiments, a dose may beadministered in two or more injections to minimize injection sitereaction in an individual.

“Dosage unit” means a form in which a pharmaceutical agent is provided.In certain embodiments, a dosage unit is a vial containing lyophilizedoligonucleotide. In certain embodiments, a dosage unit is a vialcontaining reconstituted oligonucleotide.

“Therapeutically effective amount” refers to an amount of apharmaceutical agent that provides a therapeutic benefit to an animal.

“Pharmaceutical composition” means a mixture of substances suitable foradministering to an individual that includes a pharmaceutical agent. Forexample, a pharmaceutical composition may comprise a modifiedoligonucleotide and a sterile aqueous solution.

“Pharmaceutical agent” means a substance that provides a therapeuticeffect when administered to a subject.

“Active pharmaceutical ingredient” means the substance in apharmaceutical composition that provides a desired effect.

“Metastasis” means the process by which cancer spreads from the place atwhich it first arose as a primary tumor to other locations in the body.The metastatic progression of a primary tumor reflects multiple stages,including dissociation from neighboring primary tumor cells, survival inthe circulation, and growth in a secondary location.

“Overall survival time” means the time period for which a subjectsurvives after diagnosis of or treatment for a disease. In certainembodiments, the disease is cancer.

“Progression-free survival” means the time period for which a subjecthaving a disease survives, without the disease getting worse. In certainembodiments, progression-free survival is assessed by staging or scoringthe disease. In certain embodiments, progression-free survival of asubject having liver cancer is assessed by evaluating tumor size, tumornumber, and/or metastasis.

“Blood tumor marker” means a biomarker that increases or decreases inthe blood of a subject having cancer.

“Improved liver function” means the change in liver function towardnormal limits. In certain embodiments, liver function is assessed bymeasuring molecules found in a subject's blood. For example, in certainembodiments, improved liver function is measured by a reduction in bloodliver transaminase levels.

“Acceptable safety profile” means a pattern of side effects that iswithin clinically acceptable limits.

“Side effect” means a physiological response attributable to a treatmentother than desired effects. In certain embodiments, side effectsinclude, without limitation, injection site reactions, liver functiontest abnormalities, renal function abnormalities, liver toxicity, renaltoxicity, central nervous system abnormalities, and myopathies. Suchside effects may be detected directly or indirectly. For example,increased aminotransferase levels in serum may indicate liver toxicityor liver function abnormality. For example, increased bilirubin mayindicate liver toxicity or liver function abnormality.

“Injection site reaction” means inflammation or abnormal redness of skinat a site of injection in an individual.

“Subject compliance” means adherence to a recommended or prescribedtherapy by a subject.

“Comply” means the adherence with a recommended therapy by a subject.

“Recommended therapy” means a treatment recommended by a medicalprofessional for the treatment, amelioration, or prevention of adisease.

“Target nucleic acid,” “target RNA,” “target RNA transcript” and“nucleic acid target” all mean any nucleic acid capable of beingtargeted by antisense compounds.

“Targeting” means the process of design and selection of nucleobasesequence that will hybridize to a target nucleic acid and induce adesired effect.

“Targeted to” means having a nucleobase sequence that will allowhybridization to a target nucleic acid to induce a desired effect. Incertain embodiments, a desired effect is reduction of a target nucleicacid.

“Modulation” means to a perturbation of function or activity. In certainembodiments, modulation means an increase in gene expression. In certainembodiments, modulation means a decrease in gene expression.

“Expression” means any functions and steps by which a gene's codedinformation is converted into structures present and operating in acell.

“5′ target site” refers to the nucleobase of a target nucleic acid whichis complementary to the 5′-most nucleobase of a particularoligonucleotide.

“3′ target site” means the nucleobase of a target nucleic acid which iscomplementary to the 3′-most nucleobase of a particular oligonucleotide.

“Region” means a portion of linked nucleosides within a nucleic acid. Incertain embodiments, a modified oligonucleotide has a nucleobasesequence that is complementary to a region of a target nucleic acid. Forexample, in certain such embodiments a modified oligonucleotide iscomplementary to a region of a miRNA stem-loop sequence. In certain suchembodiments, a modified oligonucleotide is fully complementary to aregion of a miRNA stem-loop sequence.

“Segment” means a smaller or sub-portion of a region.

“Nucleobase sequence” means the order of contiguous nucleobases, in a 5′to 3′ orientation, independent of any sugar, linkage, and/or nucleobasemodification.

“Contiguous nucleobases” means nucleobases immediately adjacent to eachother in a nucleic acid.

“Nucleobase complementarity” means the ability of two nucleobases topair non-covalently via hydrogen bonding.

“Complementary” means a first nucleobase sequence is at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 97%, at least 98% at least 99%, or100%, identical to the complement of a second nucleobase sequence over aregion of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 ormore nucleobases, or that the two sequences hybridize under stringenthybridization conditions.

“Complementarity” means the nucleobase pairing ability between a firstnucleic acid and a second nucleic acid.

“Fully complementary” means each nucleobase of a first nucleic acid iscapable of pairing with a nucleobase at each corresponding position in asecond nucleic acid. For example, in certain embodiments, a modifiedoligonucleotide wherein each nucleobase has complementarity to anucleobase within a region of a miRNA stem-loop sequence is fullycomplementary to the miRNA stem-loop sequence. In certain embodiments, amodified oligonucleotide that is fully complementary to a miRNA or aprecursor thereof has a nucleobase sequence that is identical to thecomplement of a miRNA or a precursor thereof over a region of 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleobases.

“Percent complementarity” means the number of complementary nucleobasesin a nucleic acid divided by the length of the nucleic acid. In certainembodiments, percent complementarity of a modified oligonucleotide meansthe number of nucleobases that are complementary to the target nucleicacid, divided by the length of the modified oligonucleotide.

“Percent identity” means the number of nucleobases in first nucleic acidthat are identical to nucleobases at corresponding positions in a secondnucleic acid, divided by the total number of nucleobases in the firstnucleic acid.

“Substantially identical” used herein may mean that a first and secondnucleobase sequence are at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 97%, at least 98% at least 99%, or 100%, identical over a regionof 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or morenucleobases.

“Hybridize” means the annealing of complementary nucleic acids thatoccurs through nucleobase complementarity.

“Mismatch” means a nucleobase of a first nucleic acid that is notcapable of pairing with a nucleobase at a corresponding position of asecond nucleic acid.

“Non-complementary nucleobase” means two nucleobases that are notcapable of pairing through hydrogen bonding.

“Identical” means having the same nucleobase sequence.

“miR-21” means the mature miRNA having the nucleobase sequence set forthin SEQ ID NO: 9.

“miR-21 stem-loop sequence” means the stem-loop sequence having thenucleobase sequence set forth in SEQ ID NO: 1.

“miR-125a-5p” means the mature miRNA having the nucleobase sequence setforth in SEQ ID NO: 10.

“miR-125a stem-loop sequence” means the stem-loop sequence having thenucleobase sequence set forth in SEQ ID NO: 2.

“miR-191” means the mature miRNA having the nucleobase sequence setforth in SEQ ID NO: 11.

“miR-191 stem-loop sequence” means the stem-loop sequence having thenucleobase sequence set forth in SEQ ID NO: 3.

“miR-210” means the mature miRNA having the nucleobase sequence setforth in SEQ ID NO: 12.

“miR-210 stem-loop sequence” means the stem-loop sequence having thenucleobase sequence set forth in SEQ ID NO: 4.

“miR-222” means the mature miRNA having the nucleobase sequence setforth in SEQ ID NO: 13.

“miR-222 stem-loop sequence” means the stem-loop sequence having thenucleobase sequence set forth in SEQ ID NO: 5.

“miR-378” means the mature miRNA having the nucleobase sequence setforth in SEQ ID NO: 14.

“miR-378 stem-loop sequence” means the stem-loop sequence having thenucleobase sequence set forth in SEQ ID NO: 6.

“miR-423-3p” means the mature miRNA having the nucleobase sequence setforth in SEQ ID NO: 15.

“miR-423 stem-loop sequence” means the stem-loop sequence having thenucleobase sequence set forth in SEQ ID NO: 7.

“miR-638” means the mature miRNA having the nucleobase sequence setforth in SEQ ID NO: 16.

“miR-638 stem-loop sequence” means the stem-loop sequence having thenucleobase sequence set forth in SEQ ID NO: 8.

“miR-181a” means the mature miRNA having the nucleobase sequence setforth in SEQ ID NO: 31.

“miR-181a*” means the mature miRNA having the nucleobase sequence setforth in SEQ ID NO: 32.

“miR-181b” means the mature miRNA having the nucleobase sequence setforth in SEQ ID NO: 33.

“miR-181a-1 stem-loop sequence” means the stem-loop sequence having thenucleobase sequence set forth in SEQ ID NO: 34.

“miR-181a-2 stem-loop sequence” means the stem-loop sequence having thenucleobase sequence set forth in SEQ ID NO: 35.

“miR-181b-1 stem-loop sequence” means the stem-loop sequence having thenucleobase sequence set forth in SEQ ID NO: 36.

“miR-181b-2 stem-loop sequence” means the stem-loop sequence having thenucleobase sequence set forth in SEQ ID NO: 37.

“miRNA” or “miR” means a non-coding RNA between 18 and 25 nucleobases inlength, which is the product of cleavage of a pre-miRNA by the enzymeDicer. Examples of mature miRNAs are found in the miRNA database knownas miRBase (microrna.sanger.ac.uk).

“Pre-miRNA” or “pre-miR” means a non-coding RNA having a hairpinstructure, which is the product of cleavage of a pri-miR by thedouble-stranded RNA-specific ribonuclease known as Drosha.

“Stem-loop sequence” means an RNA having a hairpin structure andcontaining a mature miRNA sequence. Pre-miRNA sequences and stem-loopsequences may overlap. Examples of stem-loop sequences are found in themiRNA database known as miRBase (microrna.sanger.ac.uk).

“Pri-miRNA” or “pri-miR” means a non-coding RNA having a hairpinstructure that is a substrate for the double-stranded RNA-specificribonuclease Drosha.

“miRNA precursor” means a transcript that originates from a genomic DNAand that comprises a non-coding, structured RNA comprising one or moremiRNA sequences. For example, in certain embodiments a miRNA precursoris a pre-miRNA. In certain embodiments, a miRNA precursor is apri-miRNA.

“Monocistronic transcript” means a miRNA precursor containing a singlemiRNA sequence.

“Polycistronic transcript” means a miRNA precursor containing two ormore miRNA sequences.

“Seed sequence” means nucleotides 2 to 6 or 2 to 7 from the 5′-end of amature miRNA sequence.

“Oligomeric compound” means a compound comprising a polymer of linkedmonomeric subunits.

“Oligonucleotide” means a polymer of linked nucleosides, each of whichcan be modified or unmodified, independent from one another.

“Naturally occurring internucleoside linkage” means a 3′ to 5′phosphodiester linkage between nucleosides.

“Natural sugar” means a sugar found in DNA (2′-H) or RNA (2′-OH).

“Natural nucleobase” means a nucleobase that is unmodified relative toits naturally occurring form.

“Internucleoside linkage” means a covalent linkage between adjacentnucleosides.

“Linked nucleosides” means nucleosides joined by a covalent linkage.

“Nucleobase” means a heterocyclic moiety capable of non-covalentlypairing with another nucleobase.

“Nucleoside” means a nucleobase linked to a sugar.

“Nucleotide” means a nucleoside having a phosphate group covalentlylinked to the sugar portion of a nucleoside.

“Modified oligonucleotide” means an oligonucleotide having one or moremodifications relative to a naturally occurring terminus, sugar,nucleobase, and/or internucleoside linkage.

“Single-stranded modified oligonucleotide” means a modifiedoligonucleotide which is not hybridized to a complementary strand.

“Modified internucleoside linkage” means any change from a naturallyoccurring internucleoside linkage.

“Phosphorothioate internucleoside linkage” means a linkage betweennucleosides where one of the non-bridging atoms is a sulfur atom.

“Modified sugar” means substitution and/or any change from a naturalsugar.

“Modified nucleobase” means any substitution and/or change from anatural nucleobase.

“5-methylcytosine” means a cytosine modified with a methyl groupattached to the 5′ position.

“2′-O-methyl sugar” or “2′-OMe sugar” means a sugar having a O-methylmodification at the 2′ position.

“2′-O-methoxyethyl sugar” or “2′-MOE sugar” means a sugar having a0-methoxyethyl modification at the 2′ position.

“2′-O-fluoro” or “2′-F” means a sugar having a fluoro modification ofthe 2′ position.

“Bicyclic sugar moiety” means a sugar modified by the bridging of twonon-geminal ring atoms.

“2′-O-methoxy ethyl nucleoside” means a 2′-modified nucleoside having a2′-O-methoxyethyl sugar modification.

“2′-fluoro nucleoside” means a 2′-modified nucleoside having a 2′-fluorosugar modification.

“2′-O-methyl” nucleoside means a 2′-modified nucleoside having a2′-O-methyl sugar modification.

“Bicyclic nucleoside” means a 2′-modified nucleoside having a bicyclicsugar moiety.

“Motif” means a pattern of modified and/or unmodified nucleobases,sugars, and/or internucleoside linkages in an oligonucleotide.

A “fully modified oligonucleotide” means each nucleobase, each sugar,and/or each internucleoside linkage is modified.

A “uniformly modified oligonucleotide” means each nucleobase, eachsugar, and/or each internucleoside linkage has the same modificationthroughout the modified oligonucleotide.

A “gapmer” means a modified oligonucleotide having an internal region oflinked nucleosides positioned between two external regions of linkednucleosides, where the nucleosides of the internal region comprise asugar moiety different than that of the nucleosides of each externalregion.

A “gap segment” is an internal region of a gapmer that is positionedbetween the external regions.

A “wing segment” is an external region of a gapmer that is located atthe 5′ or 3′ terminus of the internal region.

A “symmetric gapmer” means each nucleoside of each external regioncomprises the same sugar modification.

An “asymmetric gapmer” means each nucleoside of one external regioncomprises a first sugar modification, and each nucleoside of the otherexternal region comprises a second sugar modification.

A “stabilizing modification” means a modification to a nucleoside thatprovides enhanced stability to a modified oligonucleotide, in thepresence of nucleases, relative to that provided by 2′-deoxynucleosideslinked by phosphodiester internucleoside linkages. For example, incertain embodiments, a stabilizing modification is a stabilizingnucleoside modification. In certain embodiments, a stabilizingmodification is a internucleoside linkage modification.

A “stabilizing nucleoside” means a nucleoside modified to provideenhanced nuclease stability to an oligonucleotide, relative to thatprovided by a 2′-deoxynucleoside. In one embodiment, a stabilizingnucleoside is a 2′-modified nucleoside.

A “stabilizing internucleoside linkage” means an internucleoside linkagethat provides enhanced nuclease stability to an oligonucleotide relativeto that provided by a phosphodiester internucleoside linkage. In oneembodiment, a stabilizing internucleoside linkage is a phosphorothioateinternucleoside linkage.

Overview

Liver cancer is a common cause of cancer deaths in both men and womenworldwide. The incidence of hepatocellular carcinoma (HCC), the mostcommon type of liver cancer, is rising in relation to the increasingincidence of hepatitis C viral infection. Certain HCC cases have beenlinked to chronic hepatitis B infection, chronic hepatitis C infection,or cirrhosis.

Subjects with HCC have a very poor prognosis, with typical mediansurvival from the date of diagnosis ranging from 7 to 8 months, and a 5year survival rate of less than 5%. Limited treatments are available forHCC. Subjects with early stage disease may be treated by liver resectionor liver transplantation. However, in approximately 85% of subjects thedisease is too advanced at the time of diagnosis for liver resection ortransplantation. Subjects with intermediate disease may be candidatesfor chemoembolization. However, the poor health of subjects withadvanced disease limits the use of chemoembolization.

Certain changes in miRNA expression patterns in cancer cells, includingliver cancer cells such as HCC, relative to non-cancerous cells, havebeen reported. Both increases and decreases in miRNA expression havebeen described in relation to cancer. The total number of miRNAs in thehuman genome is estimated to range from approximately 500 to severalthousand. In view of this high number of total miRNAs, identification ofparticular miRNAs linked to particular cancer types is necessary inorder to identify miRNAs that could be targeted for cancer therapy,either through inhibition or augmentation of the miRNA.

Accordingly, there exists a need for the identification of miRNAs thatcan be inhibited for the treatment of liver cancer, including HCC. Alsoneeded are inhibitory agents useful for the treatment of liver cancer,such as HCC. Further, there exists a need for methods of treating livercancer, such as HCC, by administering to a subject in need thereof apharmaceutical agent capable of inhibiting a miRNA identified asdysregulated in connection with liver cancer, such as HCC. As cancer isa disease caused by the uncontrolled proliferation of cells, as well asincreased cell survival, desirable traits of pharmaceutical agents forthe treatment of liver cancer include the ability to reduce cellproliferation, and/or induce apoptosis, which will in turn reduce tumorsize, reduce tumor number, and/or prevent or slow the metastasis ofliver cancer cells.

In certain embodiments, the methods provided herein are useful for thetreatment of liver cancer, such as HCC. These methods may result in oneor more clinically desirable outcomes in a subject having liver cancer,such as reduction in tumor number and/or size, reduced metastaticprogression, prolonged survival time, and/or increased progression-freesurvival time. Also provided herein are pharmaceutical agents, such asmodified oligonucleotides, that may be used for the treatment of livercancer, such as HCC.

As illustrated herein, using microarrays containing probes designed toabout 700 miRNAs, liver samples from HCC tumors were compared to normalliver tissue samples. Of the about 700 miRNAs tested, approximately 90were found to be upregulated in HCC samples relative to normal livertissue samples. Following in vitro experiments in HCC-derived celllines, 8 miRNAs were selected as candidate miRNAs to be targeted for HCCtherapy: miR-21, miR-125a-5p, miR-191, miR-210, miR-222, miR-378,miR-423-3p, and miR-638. As illustrated herein, a reduction in cellproliferation was observed following the inhibition of 8 of these miRNAsin liver cancer cell lines, using modified oligonucleotidescomplementary to the miRNAs. Additionally, inhibition of 7 of the miRNAsresulted in increased apoptosis of liver cancer cells. As such, themodified oligonucleotides complementary to each of these 8 miRNAs arepharmaceutical agents for the treatment of liver cancer, including HCC.

As illustrated herein in a mouse subcutaneous tumor model, theadministration of a modified oligonucleotide targeted to microRNAsidentified as upregulated in HCC resulted in tumor volume reduction.Accordingly, such modified oligonucleotides are pharmaceutical agentsfor the treatment of liver cancer, including HCC.

Certain Treatments

In certain embodiments, the present invention provides methods for thetreatment of cancer comprising administering to a subject in needthereof a modified oligonucleotide complementary to a miRNA. A subjectmay be diagnosed with liver cancer following the administration ofmedical tests well-known to those in the medical profession. The livercancer may be hepatocellular carcinoma (HCC). The diagnosis ofhepatocellular carcinoma is typically made by liver imaging tests suchas abdominal ultrasound, helical computed tomography (CT) scan or triplephase CT scan. Such imaging tests may be performed in conjunction withmeasurement of blood levels of alpha-fetoprotein and/or blood levels ofdes-gamma-carboxyprothrombin. In certain subjects, MRI may be used inplace of CT scan. The liver imaging tests allow the assessment of thetumor size, number, location, metastasis outside the liver, patency andor invasion of the arteries and veins of the liver by the tumor. Thisassessment aids the decision as to the mode of therapeutic or palliativeintervention that is appropriate. The final diagnosis is typicallyconfirmed by needle biopsy and histopathological examination.

Accordingly, in certain embodiments, the liver cancer is detectedfollowing a computed tomography (CT) scan that detects tumors. Incertain embodiments, the liver cancer is detected following magneticresonance imaging (MRI). In certain embodiments, HCC is characterized asa single primary tumor. In certain embodiments, HCC is characterized asmultiple primary tumors. In certain embodiments, HCC is characterized asa poorly defined primary tumor with an infiltrative growth pattern. Incertain embodiments, the HCC is a single primary tumor with vascularinvasion. In certain embodiments, the HCC is characterized as multipleprimary tumors with vascular invasion. In certain embodiments, the HCChas metastasized to one or more lymph nodes. In certain suchembodiments, the lymph nodes are regional lymph nodes. In certainembodiments, the HCC has metastasized to one or more distant tissues. Incertain embodiments, the HCC has metastasized to other regions of theliver, the portal vein, lymph nodes, adrenal glands, bone or lungs. Incertain embodiments, fibrosis is present.

A number of systems have been employed to predict the prognosis for HCC,including the TNM system, the Okuda system, the Barcelona Clinic LiverCancer (BCLC) and the CLIP score. Each of these systems incorporatesfour features that have been recognized as being important determinantsof survival: the severity of underlying liver disease, the size of thetumor, extension of the tumor into adjacent structures, and the presenceof metastases. The TNM system classifies HCC as stage I, II, III, IV, orV. The BCLC classifies HCC as Stage A1, A2, A3, A4, B, C, and D, andincludes consideration of a Child-Pugh score.

In certain embodiments, liver cancer is classified as Stage 1, Stage 2,Stage 3A, Stage 3B, Stage 3C, or Stage 4. Stage 1 is characterized by acancer is no bigger than 2 cm in size and that has not begun to spread.At Stage 2, the cancer is affecting blood vessels in the liver, or thereis more than one tumor in the liver. At Stage 3A, the cancer is biggerthan 5 cm in size or has spread to the blood vessels near the liver. AtStage 3B, the cancer has spread to nearby organs, such as the bowel orthe stomach, but has not spread to the lymph nodes. At Stage 3C thecancer can be of any size and has spread to nearby lymph nodes. At Stage4 the cancer has spread to parts of the body further away from theliver, such as the lungs.

Biomarkers in a subject's blood may be used to augment a diagnosis ofliver cancer, stage a liver cancer, or develop a prognosis for survival.Such biomarkers include blood tumor biomarkers, such asalpha-fetoprotein and des-gamma carboxyprothrombin. In certain suchembodiments, the subject has elevated blood alpha-fetoprotein. Incertain such embodiments, the subject has elevated blood des-gammacarboxyprothrombin.

A subject having liver cancer may also suffer from abnormal liverfunction. Liver function may be assessed by liver function tests, whichmeasure, among other things, blood levels of liver transaminases. Incertain embodiments, a subject having abnormal liver function haselevated blood liver transaminases. Blood liver transaminases includealanine aminotransferase (ALT) and aspartate aminotransferase (AST). Incertain embodiments, a subject having abnormal liver function haselevated blood bilirubin. In certain embodiments, a subject has abnormalblood albumin levels.

In certain embodiments, a subject's liver function is assessed by theChild-Pugh classification system, which defines three classes of liverfunction. In this classification system, points are assigned tomeasurements in one of five categories: bilirubin levels, albuminlevels, prothrombin time, ascites, and encephalopathy. One point isassigned per each of the following characteristics present: bloodbilirubin of less than 2.0 mg/dl; blood albumin of greater than 3.5mg/dl; a prothrombin time of less than 1.7 international normalizedratio (INR); ascites is absent; or encephalopathy is absent. Two pointsare assigned per each of the following characteristics present: bloodbilirubin of 2-3 mg/dl; blood bilirubin of 3.5 to 2.8 mg/dl; prothrombintime of 1.7-2.3 INR; ascites is mild to moderate; or encephalopathy ismild. Three points are assigned per each of the followingcharacteristics present: bilirubin of greater than 3.0 mg/dl; bloodalbumin of less than 2.8 mg/dl; prothrombin time of greater than 2.3INR; ascites is severe to refractory; or encephalopathy is severe. Thescores are added and Class A is assigned for a score of 5-6 points,Class B is assigned for a score of 7-9 points, and Class C is assignedfor a score of 10-15 points,

A subject having liver cancer may have suffered from chronic hepatitis Cinfection, chronic hepatitis B infection, or cirrhosis. Subjects havingliver cancer caused by hepatitis C infection, hepatitis B infection, orcirrhosis may be treated by the methods described herein.

A subject's response to treatment may be evaluated by tests similar tothose used to diagnosis the liver cancer, including, without limitation,CT scan, MRI, and needle biopsy. Response to treatment may also beassessed by measuring biomarkers in blood, for comparison topre-treatment levels of biomarkers.

Administration of a pharmaceutical composition of the present inventionto a subject having liver cancer results in one or more clinicallydesirable outcomes. Such clinically desirable outcomes include reductionof tumor number or reduction of tumor size. Additional clinicallydesirable outcomes include the extension of overall survival time of thesubject, and/or extension of progression-free survival time of thesubject. In certain embodiments, administration of a pharmaceuticalcomposition of the invention prevents an increase in tumor size and/ortumor number. In certain embodiments, administration of a pharmaceuticalcomposition of the invention prevents metastatic progression. In certainembodiments, administration of a pharmaceutical composition of theinvention slows or stops metastatic progression. In certain embodiments,administration of a pharmaceutical composition of the invention preventsthe recurrence of liver tumors. In certain embodiments, administrationof a pharmaceutical composition of the invention prevents recurrence ofliver tumor metastasis. In certain embodiments, administration of apharmaceutical composition of the invention prevents the recurrence ofHCC-derived tumors. In certain embodiments, administration of apharmaceutical composition of the invention prevents the recurrence ofHCC-derived tumor metastasis.

Administration of a pharmaceutical composition of the present inventionto liver cancer cells, including HCC cells, may result in desirablephenotypic effects. In certain embodiments, a modified oligonucleotidemay stop, slow or reduce the uncontrolled proliferation of liver cancercells. In certain embodiments, a modified oligonucleotide may induceapoptosis in liver cancer cells. In certain embodiments, a modifiedoligonucleotide may reduce liver cancer cell survival.

A miRNA hybridizes to a mRNA to regulate expression of the mRNA and itsprotein product. Generally, the hybridization of a miRNA to its mRNAtarget inhibits expression of the mRNA. Thus, the inhibition of a miRNAmay result in the increased expression of a miRNA nucleic acid target.In certain embodiments, the inhibition of a miRNA results in theincrease of a protein encoded by a miRNA nucleic acid target. Forexample, in certain embodiments, the antisense inhibition of miR-222results in an increase of p27^(kip1).

Certain desirable clinical outcomes may be assessed by measurements ofblood biomarkers. In certain embodiments, administration of apharmaceutical composition of the invention may result in the decreaseof blood alpha-fetoprotein and/or blood des-gamma carboxyprothrombin.Administration of a pharmaceutical composition of the invention mayfurther result in the improvement of liver function, as evidenced by areduction in blood ALT and/or AST levels.

Certain Compounds

The compounds provided herein are useful for the treatment of livercancer, such as HCC. In certain embodiments, the compound comprises amodified oligonucleotide. In certain such embodiments, the compoundconsists of a modified oligonucleotide.

In certain such embodiments, the compound comprises a modifiedoligonucleotide hybridized to a complementary strand, i.e. the compoundcomprises a double-stranded oligomeric compound. In certain embodiments,the hybridization of a modified oligonucleotide to a complementarystrand forms at least one blunt end. In certain such embodiments, thehybridization of a modified oligonucleotide to a complementary strandforms a blunt end at each terminus of the double-stranded oligomericcompound. In certain embodiments, a terminus of a modifiedoligonucleotide comprises one or more additional linked nucleosidesrelative to the number of linked nucleosides of the complementarystrand. In certain embodiments, the one or more additional nucleosidesare at the 5′ terminus of a modified oligonucleotide. In certainembodiments, the one or more additional nucleosides are at the 3′terminus of a modified oligonucleotide. In certain embodiments, at leastone nucleobase of a nucleoside of the one or more additional nucleosidesis complementary to the target RNA. In certain embodiments, eachnucleobase of each one or more additional nucleosides is complementaryto the target RNA. In certain embodiments, a terminus of thecomplementary strand comprises one or more additional linked nucleosidesrelative to the number of linked nucleosides of a modifiedoligonucleotide. In certain embodiments, the one or more additionallinked nucleosides are at the 3′ terminus of the complementary strand.In certain embodiments, the one or more additional linked nucleosidesare at the 5′ terminus of the complementary strand. In certainembodiments, two additional linked nucleosides are linked to a terminus.In certain embodiments, one additional nucleoside is linked to aterminus.

In certain embodiments, the compound comprises a modifiedoligonucleotide conjugated to one or more moieties which enhance theactivity, cellular distribution or cellular uptake of the resultingantisense oligonucleotides. In certain such embodiments, the moiety is acholesterol moiety or a lipid moiety. Additional moieties forconjugation include carbohydrates, phospholipids, biotin, phenazine,folate, phenanthridine, anthraquinone, acridine, fluoresceins,rhodamines, coumarins, and dyes. In certain embodiments, a conjugategroup is attached directly to a modified oligonucleotide. In certainembodiments, a conjugate group is attached to a modified oligonucleotideby a linking moiety selected from amino, hydroxyl, carboxylic acid,thiol, unsaturations (e.g., double or triple bonds),8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), 6-aminohexanoic acid (AHEX or AHA),substituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl,and substituted or unsubstituted C2-C10 alkynyl. In certain suchembodiments, a substituent group is selected from hydroxyl, amino,alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen,alkyl, aryl, alkenyl and alkynyl.

In certain such embodiments, the compound comprises a modifiedoligonucleotide having one or more stabilizing groups that are attachedto one or both termini of a modified oligonucleotide to enhanceproperties such as, for example, nuclease stability. Included instabilizing groups are cap structures. These terminal modificationsprotect a modified oligonucleotide from exonuclease degradation, and canhelp in delivery and/or localization within a cell. The cap can bepresent at the 5′-terminus (5′-cap), or at the 3′-terminus (3′-cap), orcan be present on both termini. Cap structures include, for example,inverted deoxy abasic caps.

Suitable cap structures include a 4′,5′-methylene nucleotide, a1-(beta-D-erythrofuranosyl) nucleotide, a 4′-thio nucleotide, acarbocyclic nucleotide, a 1,5-anhydrohexitol nucleotide, anL-nucleotide, an alpha-nucleotide, a modified base nucleotide, aphosphorodithioate linkage, a threo-pentofuranosyl nucleotide, anacyclic 3′,4′-seco nucleotide, an acyclic 3,4-dihydroxybutyl nucleotide,an acyclic 3,5-dihydroxypentyl nucleotide, a 3′-3′-inverted nucleotidemoiety, a 3′-3′-inverted abasic moiety, a 3′-2′-inverted nucleotidemoiety, a 3′-2′-inverted abasic moiety, a 1,4-butanediol phosphate, a3′-phosphoramidate, a hexylphosphate, an aminohexyl phosphate, a3′-phosphate, a 3′-phosphorothioate, a phosphorodithioate, a bridgingmethylphosphonate moiety, and a non-bridging methylphosphonate moiety5′-amino-alkyl phosphate, a 1,3-diamino-2-propyl phosphate,3-aminopropyl phosphate, a 6-aminohexyl phosphate, a 1,2-aminododecylphosphate, a hydroxypropyl phosphate, a 5′-5′-inverted nucleotidemoiety, a 5′-5′-inverted abasic moiety, a 5′-phosphoramidate, a5′-phosphorothioate, a 5′-amino, a bridging and/or non-bridging5′-phosphoramidate, a phosphorothioate, and a 5′-mercapto moiety.

Certain Nucleobase Sequences

In certain embodiments, a modified oligonucleotide has a sequence thatis complementary to a miRNA or a precursor thereof. Nucleobase sequencesof mature miRNAs and their corresponding stem-loop sequences describedherein are the sequences found in miRBase, an online searchable databaseof miRNA sequences and annotation, found at microrna.sanger.ac.uk.Entries in the miRBase Sequence database represent a predicted hairpinportion of a miRNA transcript (the stem-loop), with information on thelocation and sequence of the mature miRNA sequence. The miRNA stem-loopsequences in the database are not strictly precursor miRNAs(pre-miRNAs), and may in some instances include the pre-miRNA and someflanking sequence from the presumed primary transcript. The miRNAnucleobase sequences described herein encompass any version of themiRNA, including the sequences described in Release 10.0 of the miRBasesequence database and sequences described in any earlier Release of themiRBase sequence database. A sequence database release may result in there-naming of certain miRNAs. For example, miR-378 of Release 10.0described herein was formerly named miR-422b. A sequence databaserelease may result in a variation of a mature miRNA sequence. Forexample, miR-125a-5p of Release 10.0 is found at nucleobases 15-38 ofthe miR-125a stem-loop sequence (SEQ ID NO: 2). miR-125a in a previousdatabase Releases is found at nucleobases 15-37 of the miR-125astem-loop sequence (SEQ ID NO: 2). The compositions of the presentinvention encompass modified oligonucleotides that are complementary toany nucleobase sequence version of the miRNAs described herein.

In certain embodiments, a modified oligonucleotide has a nucleobasesequence that is complementary to a miRNA or a precursor thereof,meaning that the nucleobase sequence of a modified oligonucleotide is aleast 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identicalto the complement of a miRNA or precursor thereof over a region of 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleobases,or that the two sequences hybridize under stringent hybridizationconditions. Accordingly, in certain embodiments the nucleobase sequenceof a modified oligonucleotide may have one or more mismatched basepairswith respect to its target miRNA or precursor sequence, and is capableof hybridizing to its target sequence. In certain embodiments, amodified oligonucleotide has a nucleobase sequence that is fullycomplementary to a miRNA or precursor thereof, meaning that thenucleobase sequence of a modified oligonucleotide is 100% identical ofthe complement of an miRNA or a precursor thereof over a region of 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleobases.

In certain embodiments, a modified oligonucleotide has a sequence thatis complementary to a nucleobase sequence of a miRNA stem-loop sequenceselected from the miR-21 stem-loop sequence (SEQ ID NO: 1), the miR-125astem-loop sequence (SEQ ID NO: 2), the miR-191 stem-loop sequence (SEQID NO: 3), the miR-210 stem-loop sequence (SEQ ID NO: 4), the miR-222stem-loop sequence (SEQ ID NO: 5), the miR-378 stem-loop sequence (SEQID NO: 6), the miR-423 stem-loop sequence (SEQ ID NO: 7), and themiR-638 stem-loop sequence (SEQ ID NO: 8).

In certain embodiments, a modified oligonucleotide has a sequence thatis complementary to a nucleobase sequence of a miRNA, where thenucleobase sequence of the miRNA is selected from SEQ ID NO: 9, 10, 11,12, 13, 14, 15, and 16.

In certain embodiments, a modified oligonucleotide has a nucleobasesequence that is complementary to a region of the miR-21 stem-loopsequence (SEQ ID NO: 1). In certain embodiments, a modifiedoligonucleotide has a nucleobase sequence that is complementary to theregion of nucleobases 8-29 of SEQ ID NO: 1. In certain embodiments, amodified oligonucleotide has a nucleobase sequence that is complementaryto the nucleobase sequence of miR-21 (SEQ ID NO: 9). In certainembodiments, a modified oligonucleotide has a nucleobase sequence thatis complementary to nucleobases 1-22 of SEQ ID NO: 9. In certainembodiments, a modified oligonucleotide has a nucleobase sequencecomprising the nucleobase sequence TCAACATCAGTCTGATAAGCTA (SEQ ID NO:17). In certain embodiments, a modified oligonucleotide has a nucleobasesequence consisting of the nucleobase sequence TCAACATCAGTCTGATAAGCTA(SEQ ID NO: 17).

In certain embodiments, a modified oligonucleotide has a nucleobasesequence that is complementary to a region of the miR-125a stem-loopsequence (SEQ ID NO: 2). In certain embodiments, a modifiedoligonucleotide has a nucleobase sequence that is complementary to theregion of nucleobases 15-37 of SEQ ID NO: 2. In certain embodiments, amodified oligonucleotide has a nucleobase sequence that is complementaryto the region of nucleobases 15-38 of SEQ ID NO: 2. In certainembodiments, a modified oligonucleotide has a nucleobase sequence thatis complementary to the nucleobase sequence of miR-125-5p (SEQ ID NO:10). In certain embodiments, a modified oligonucleotide has a nucleobasesequence that is complementary to nucleobases 1-23 of SEQ ID NO: 10. Incertain embodiments, a modified oligonucleotide has a nucleobasesequence that is complementary to nucleobases 1-24 of SEQ ID NO: 10. Incertain embodiments, a modified oligonucleotide has a nucleobasesequence comprising the nucleobase sequence CACAGGTTAAAGGGTCTCAGGGA (SEQID NO: 18). In certain embodiments, a modified oligonucleotide has anucleobase sequence consisting of the nucleobase sequenceCACAGGTTAAAGGGTCTCAGGGA (SEQ ID NO: 18). In certain embodiments, amodified oligonucleotide has a nucleobase sequence consisting of thenucleobase sequence TCACAGGTTAAAGGGTCTCAGGGA (SEQ ID NO: 19).

In certain embodiments, a modified oligonucleotide has a nucleobasesequence that is complementary to a region of the miR-191 stem-loopsequence (SEQ ID NO: 3). In certain embodiments, a modifiedoligonucleotide has a nucleobase sequence that is complementary to theregion of nucleobases 16-37 of SEQ ID NO: 3. In certain embodiments, amodified oligonucleotide has a nucleobase sequence that is complementaryto the region of nucleobases 16-38 of SEQ ID NO: 3. In certainembodiments, a modified oligonucleotide has a nucleobase sequence thatis complementary to the nucleobase sequence of miR-191 (SEQ ID NO: 11).In certain embodiments, a modified oligonucleotide has a nucleobasesequence comprising the nucleobase sequence AGCTGCTTTTGGGATTCCGTTG (SEQID NO: 20). In certain embodiments, a modified oligonucleotide has anucleobase sequence consisting of the nucleobase sequenceAGCTGCTTTTGGGATTCCGTTG (SEQ ID NO: 20). In certain embodiments, amodified oligonucleotide has a nucleobase sequence consisting of thenucleobase sequence of CAGCTGCTTTTGGGATTCCGTTG (SEQ ID NO: 21).

In certain embodiments, a modified oligonucleotide has a nucleobasesequence that is complementary to a region of the miR-210 stem-loopsequence (SEQ ID NO: 4). In certain embodiments, a modifiedoligonucleotide has a nucleobase sequence that is complementary to theregion of nucleobases 66-87 of SEQ ID NO: 4. In certain embodiments, amodified oligonucleotide has a nucleobase sequence that is complementaryto the nucleobase sequence of miR-210 (SEQ ID NO: 12). In certainembodiments, a modified oligonucleotide has a nucleobase sequencecomprising the nucleobase sequence TCAGCCGCTGTCACACGCACAG (SEQ ID NO:22). In certain embodiments, a modified oligonucleotide has a nucleobasesequence consisting of the nucleobase sequence TCAGCCGCTGTCACACGCACAG(SEQ ID NO: 22).

In certain embodiments, a modified oligonucleotide has a nucleobasesequence that is complementary to a region of the miR-222 stem-loopsequence (SEQ ID NO: 5). In certain embodiments, a modifiedoligonucleotide has a nucleobase sequence that is complementary to theregion of nucleobases 69-89 of SEQ ID NO: 5. In certain embodiments, amodified oligonucleotide has a nucleobase sequence that is complementaryto the region of nucleobases 69-91 of SEQ ID NO: 5. In certainembodiments, a modified oligonucleotide has a nucleobase sequence thatis complementary to the nucleobase sequence of miR-222 (SEQ ID NO: 13).In certain embodiments, a modified oligonucleotide has a nucleobasesequence comprising the nucleobase sequence ACCCAGTAGCCAGATGTAGCT (SEQID NO: 24). In certain embodiments, a modified oligonucleotide has anucleobase sequence consisting of the nucleobase sequenceACCCAGTAGCCAGATGTAGCT (SEQ ID NO: 24). In certain embodiments, amodified oligonucleotide has a nucleobase sequence consisting of thenucleobase sequence GAGACCCAGTAGCCAGATGTAGCT (SEQ ID NO: 23).

In certain embodiments, a modified oligonucleotide has a nucleobasesequence that is complementary to a region of the miR-378 stem-loopsequence (SEQ ID NO: 6). In certain embodiments, a modifiedoligonucleotide has a nucleobase sequence that is complementary to theregion of nucleobases 43-63 of SEQ ID NO: 6. In certain embodiments, amodified oligonucleotide has a nucleobase sequence that is complementaryto the region of nucleobases 44-65 of SEQ ID NO: 6. In certainembodiments, a modified oligonucleotide has a nucleobase sequence thatis complementary to the nucleobase sequence of miR-378 (SEQ ID NO: 14).In certain embodiments, a modified oligonucleotide has a nucleobasesequence comprising the nucleobase sequence CCTTCTGACTCCAAGTCCAG (SEQ IDNO: 25). In certain embodiments, a modified oligonucleotide has anucleobase sequence consisting of the nucleobase sequenceGGCCTTCTGACTCCAAGTCCAG (SEQ ID NO: 26). In certain embodiments, amodified oligonucleotide has a nucleobase sequence consisting of thenucleobase sequence CCTTCTGACTCCAAGTCCAGT (SEQ ID NO: 27).

In certain embodiments, a modified oligonucleotide has a nucleobasesequence that is complementary to a region of the miR-423 stem-loopsequence (SEQ ID NO: 7). In certain embodiments, a modifiedoligonucleotide has a nucleobase sequence that is complementary to theregion of nucleobases 53-75 of SEQ ID NO: 7. In certain embodiments, amodified oligonucleotide has a nucleobase sequence that is complementaryto the region of nucleobases 53-74 of SEQ ID NO: 7. In certainembodiments, a modified oligonucleotide has a nucleobase sequence thatis complementary to the nucleobase sequence of miR-423-3p (SEQ ID NO:15). In certain embodiments, a modified oligonucleotide has a nucleobasesequence comprising the nucleobase sequence CTGAGGGGCCTCAGACCGAGCT (SEQID NO: 28).

In certain embodiments, a modified oligonucleotide has a nucleobasesequence consisting of the nucleobase sequence CTGAGGGGCCTCAGACCGAGCT(SEQ ID NO: 28). In certain embodiments, a modified oligonucleotide hasa nucleobase sequence consisting of the nucleobase sequenceTCTGAGGGGCCTCAGACCGAGCT (SEQ ID NO: 29).

In certain embodiments, a modified oligonucleotide has a nucleobasesequence that is complementary to a region of the miR-638 stem-loopsequence (SEQ ID NO: 8). In certain embodiments, a modifiedoligonucleotide has a nucleobase sequence that is complementary to theregion of nucleobases 16-40 of SEQ ID NO: 8. In certain embodiments, amodified oligonucleotide has a nucleobase sequence that is complementaryto the nucleobase sequence of miR-638 (SEQ ID NO: 16). In certainembodiments, a modified oligonucleotide has a nucleobase sequencecomprising the nucleobase sequence AGGCCGCCACCCGCCCGCGATCCCT (SEQ ID NO:30). In certain embodiments, a modified oligonucleotide has a nucleobasesequence consisting of the nucleobase sequence AGGCCGCCACCCGCCCGCGATCCCT(SEQ ID NO: 30).

In certain embodiments, a modified oligonucleotide has a nucleobasesequence that is complementary to a region of the miR-181a-1 stem-loopsequence (SEQ ID NO: 34). In certain embodiments, a modifiedoligonucleotide has a nucleobase sequence that is complementary to theregion of nucleobases 24-46 of SEQ ID NO: 34. In certain embodiments, amodified oligonucleotide has a nucleobase sequence that is complementaryto a region of the miR-181a-2 stem-loop sequence (SEQ ID NO: 35). Incertain embodiments, a modified oligonucleotide has a nucleobasesequence that is complementary to the region of nucleobases 39-61 of SEQID NO: 35. In certain embodiments, a modified oligonucleotide has anucleobase sequence that is complementary to the nucleobase sequence ofmiR-181a (SEQ ID NO: 31). In certain embodiments, a modifiedoligonucleotide has a nucleobase sequence comprising the nucleobasesequence TCACTCCGTCTGCGAAGTTAGAA (SEQ ID NO: 38).

In certain embodiments, a modified oligonucleotide has a nucleobasesequence consisting of the nucleobase sequence TCACTCCGTCTGCGAAGTTAGAA(SEQ ID NO: 38).

In certain embodiments, a modified oligonucleotide has a nucleobasesequence that is complementary to a region of the miR-181a-1 stem-loopsequence (SEQ ID NO: 34). In certain embodiments, a modifiedoligonucleotide has a nucleobase sequence that is complementary to theregion of nucleobases 64-85 of SEQ ID NO: 34. In certain embodiments, amodified oligonucleotide has a nucleobase sequence that is complementaryto a region of the miR-181a-2 stem-loop sequence (SEQ ID NO: 35). Incertain embodiments, a modified oligonucleotide has a nucleobasesequence that is complementary to the region of nucleobases 77-98 of SEQID NO: 35. In certain embodiments, a modified oligonucleotide has anucleobase sequence that is complementary to the nucleobase sequence ofmiR-181a* (SEQ ID NO: 32). In certain embodiments, a modifiedoligonucleotide has a nucleobase sequence comprising the nucleobasesequence GGATCTTACTTCGGACGTAGGA (SEQ ID NO: 39).

In certain embodiments, a modified oligonucleotide has a nucleobasesequence consisting of the nucleobase sequence GGATCTTACTTCGGACGTAGGA(SEQ ID NO: 39).

In certain embodiments, a modified oligonucleotide has a nucleobasesequence that is complementary to a region of the miR-181b-1 stem-loopsequence (SEQ ID NO: 36). In certain embodiments, a modifiedoligonucleotide has a nucleobase sequence that is complementary to theregion of nucleobases 36-58 of SEQ ID NO: 36. In certain embodiments, amodified oligonucleotide has a nucleobase sequence that is complementaryto a region of the miR-181b-2 stem-loop sequence (SEQ ID NO: 37). Incertain embodiments, a modified oligonucleotide has a nucleobasesequence that is complementary to the region of nucleobases 16-38 of SEQID NO: 37. In certain embodiments, a modified oligonucleotide has anucleobase sequence that is complementary to the nucleobase sequence ofmiR-181b (SEQ ID NO: 33). In certain embodiments, a modifiedoligonucleotide has a nucleobase sequence comprising the nucleobasesequence TCCCTCCGTCTGCTTAGTTAGAA (SEQ ID NO: 40).

In certain embodiments, a modified oligonucleotide has a nucleobasesequence consisting of the nucleobase sequence TCCCTCCGTCTGCTTAGTTAGAA(SEQ ID NO: 40).

In certain embodiments, a modified oligonucleotide has a nucleobasesequence that is complementary to a nucleobase sequence of a pre-miRsequence comprising a mature miRNA selected from miR-21, miR-125a-5p,miR-191, miR-210, miR-222, miR-378, miR-423-3p, and miR-638.

In certain embodiments, a modified oligonucleotide has a nucleobasesequence that is complementary to a nucleobase sequence of a pri-miRsequence comprising a mature miRNA selected from miR-21, miR-125a-5p,miR-191, miR-210, miR-222, miR-378, miR-423-3p, and miR-638.

In certain embodiments, a modified oligonucleotide has a nucleobasesequence that is complementary to a nucleobase sequence of a pri-miRsequence comprising a mature miRNA selected from miR-181a, miR-181a*,and miR-181b. In certain embodiments, a modified oligonucleotide has anucleobase sequence that is complementary to a nucleobase sequence of apre-miR sequence comprising a mature miRNA selected from miR-181a,miR-181a*, and miR-181b.

In certain embodiments, a modified oligonucleotide has a nucleobasesequence that is complementary to a nucleobase sequence having at least80% identity to a nucleobase sequence of a miR stem-loop sequenceselected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, and 8. In certainembodiments, a modified oligonucleotide has a nucleobase sequence thatis complementary to a nucleobase sequence having at least 85%, at least90%, at least 92%, at least 94%, at least 96%, at least 98% identity, or100% identity to a nucleobase sequence of a miR stem-loop sequenceselected from SEQ ID NOs 1, 2, 3, 4, 5, 6, 7, and 8.

In certain embodiments, a modified oligonucleotide has a nucleobasesequence that is complementary to a nucleobase sequence having at least80% identity to a nucleobase sequence of a miRNA having a nucleobasesequence selected from SEQ ID NO: 9, 10, 11, 12, 13, 14, 15, and 16. Incertain embodiments, a modified oligonucleotide has a nucleobasesequence that is complementary to a nucleobase sequence having at least85%, at least 90%, at least 92%, at least 94%, at least 96%, at least98% identity, or 100% identity to a nucleobase sequence of a miRNAnucleobase sequence selected from SEQ ID NOs 9, 10, 11, 12, 13, 14, 15,and 16.

In certain embodiments, a modified oligonucleotide has a nucleobasesequence that is complementary to a nucleobase sequence having at least80% identity to a nucleobase sequence of a miR stem-loop sequenceselected from SEQ ID NO: 34, 35, 36, and 37. In certain embodiments, amodified oligonucleotide has a nucleobase sequence that is complementaryto a nucleobase sequence having at least 85%, at least 90%, at least92%, at least 94%, at least 96%, at least 98% identity, or 100% identityto a nucleobase sequence of a miR stem-loop sequence selected from SEQID NOs 34, 35, 36, and 37.

In certain embodiments, a modified oligonucleotide has a nucleobasesequence that is complementary to a nucleobase sequence having at least80% identity to a nucleobase sequence of a miRNA having a nucleobasesequence selected from SEQ ID NO: 31, 32, and 33. In certainembodiments, a modified oligonucleotide has a nucleobase sequence thatis complementary to a nucleobase sequence having at least 85%, at least90%, at least 92%, at least 94%, at least 96%, at least 98% identity, or100% identity to a nucleobase sequence of a miRNA nucleobase sequenceselected from SEQ ID NOs 31, 32, and 33.

In certain embodiments, a nucleobase sequence of a modifiedoligonucleotide is fully complementary to a miRNA nucleobase sequencelisted herein, or a precursor thereof. In certain embodiments, amodified oligonucleotide has a nucleobase sequence having one mismatchwith respect to the nucleobase sequence of the mature miRNA, or aprecursor thereof. In certain embodiments, a modified oligonucleotidehas a nucleobase sequence having two mismatches with respect to thenucleobase sequence of the miRNA, or a precursor thereof. In certainsuch embodiments, a modified oligonucleotide has a nucleobase sequencehaving no more than two mismatches with respect to the nucleobasesequence of the mature miRNA, or a precursor thereof. In certain suchembodiments, the mismatched nucleobases are contiguous. In certain suchembodiments, the mismatched nucleobases are not contiguous.

In certain embodiments, a modified oligonucleotide consists of a numberof linked nucleosides that is equal to the length of the mature miR towhich it is complementary.

In certain embodiments, the number of linked nucleosides of a modifiedoligonucleotide is less than the length of the mature miRNA to which itis complementary. In certain such embodiments, the number of linkednucleosides of a modified oligonucleotide is one less than the length ofthe mature miR to which it is complementary. In certain suchembodiments, a modified oligonucleotide has one less nucleoside at the5′ terminus. In certain such embodiments, a modified oligonucleotide hasone less nucleoside at the 3′ terminus. In certain such embodiments, amodified oligonucleotide has two fewer nucleosides at the 5′ terminus.In certain such embodiments, a modified oligonucleotide has two fewernucleosides at the 3′ terminus. A modified oligonucleotide having anumber of linked nucleosides that is less than the length of the miRNA,wherein each nucleobase of a modified oligonucleotide is complementaryto each nucleobase at a corresponding position in a miRNA, is consideredto be a modified oligonucleotide having a nucleobase sequence that isfully complementary to a portion of a miRNA sequence.

In certain embodiments, the number of linked nucleosides of a modifiedoligonucleotide is greater than the length of the miRNA to which it iscomplementary. In certain such embodiments, the nucleobase of anadditional nucleoside is complementary to a nucleobase of a miRNAstem-loop sequence. In certain embodiments, the number of linkednucleosides of a modified oligonucleotide is one greater than the lengthof the miRNA to which it is complementary. In certain such embodiments,the additional nucleoside is at the 5′ terminus of a modifiedoligonucleotide. In certain such embodiments, the additional nucleosideis at the 3′ terminus of a modified oligonucleotide. In certainembodiments, the number of linked nucleosides of a modifiedoligonucleotide is two greater than the length of the miRNA to which itis complementary. In certain such embodiments, the two additionalnucleosides are at the 5′ terminus of a modified oligonucleotide. Incertain such embodiments, the two additional nucleosides are at the 3′terminus of a modified oligonucleotide. In certain such embodiments, oneadditional nucleoside is located at the 5′ terminus and one additionalnucleoside is located at the 3′ terminus of a modified oligonucleotide.

In certain embodiments, a portion of the nucleobase sequence of amodified oligonucleotide is fully complementary to the nucleobasesequence of the miRNA, but the entire modified oligonucleotide is notfully complementary to the miRNA. In certain such embodiments, thenumber of nucleosides of a modified oligonucleotide having a fullycomplementary portion is greater than the length of the miRNA. Forexample, a modified oligonucleotide consisting of 24 linked nucleosides,where the nucleobases of nucleosides 1 through 23 are each complementaryto a corresponding position of a miRNA that is 23 nucleobases in length,has a 23 nucleoside portion that is fully complementary to thenucleobase sequence of the miRNA and approximately 96% overallcomplementarity to the nucleobase sequence of the miRNA.

In certain embodiments, the nucleobase sequence of a modifiedoligonucleotide is fully complementary to a portion of the nucleobasesequence of a miRNA. For example, a modified oligonucleotide consistingof 22 linked nucleosides, where the nucleobases of nucleosides 1 through22 are each complementary to a corresponding position of a miRNA that is23 nucleobases in length, is fully complementary to a 22 nucleobaseportion of the nucleobase sequence of a miRNA. Such a modifiedoligonucleotide has approximately 96% overall complementarity to thenucleobase sequence of the entire miRNA, and has 100% complementarity toa 22 nucleobase portion of the miRNA.

In certain embodiments, a portion of the nucleobase sequence of amodified oligonucleotide is fully complementary to a portion of thenucleobase sequence of a miRNA, or a precursor thereof. In certain suchembodiments, 15 contiguous nucleobases of a modified oligonucleotide areeach complementary to 15 contiguous nucleobases of a miRNA, or aprecursor thereof. In certain such embodiments, 16 contiguousnucleobases of a modified oligonucleotide are each complementary to 16contiguous nucleobases of a miRNA, or a precursor thereof. In certainsuch embodiments, 17 contiguous nucleobases of a modifiedoligonucleotide are each complementary to 17 contiguous nucleobases of amiRNA, or a precursor thereof. In certain such embodiments, 18contiguous nucleobases of a modified oligonucleotide are eachcomplementary to 18 contiguous nucleobases of a miRNA, or a precursorthereof. In certain such embodiments, 19 contiguous nucleobases of amodified oligonucleotide are each complementary to 19 contiguousnucleobases of a miRNA, or a precursor thereof. In certain suchembodiments, 20 contiguous nucleobases of a modified oligonucleotide areeach complementary to 20 contiguous nucleobases of a miRNA, or aprecursor thereof. In certain such embodiments, 22 contiguousnucleobases of a modified oligonucleotide are each complementary to 22contiguous nucleobases of a miRNA, or a precursor thereof. In certainsuch embodiments, 23 contiguous nucleobases of a modifiedoligonucleotide are each complementary to 23 contiguous nucleobases of amiRNA, or a precursor thereof. In certain such embodiments, 24contiguous nucleobases of a modified oligonucleotide are eachcomplementary to 24 contiguous nucleobases of a miRNA, or a precursorthereof.

The nucleobase sequences set forth herein, including but not limited tothose found in the Examples and in the sequence listing, are independentof any modification to the nucleic acid. As such, nucleic acids definedby a SEQ ID NO may comprise, independently, one or more modifications toone or more sugar moieties, to one or more internucleoside linkages,and/or to one or more nucleobases.

Although the sequence listing accompanying this filing identifies eachnucleobase sequence as either “RNA” or “DNA” as required, in reality,those sequences may be modified with any combination of chemicalmodifications. One of skill in the art will readily appreciate that suchdesignation as “RNA” or “DNA” to describe modified oligonucleotides issomewhat arbitrary. For example, an oligonucleotide comprising anucleoside comprising a 2′-OH sugar moiety and a thymine base could bedescribed as a DNA having a modified sugar (2′-OH for the natural 2′-Hof DNA) or as an RNA having a modified base (thymine (methylated uracil)for natural uracil of RNA).

Accordingly, nucleic acid sequences provided herein, including, but notlimited to those in the sequence listing, are intended to encompassnucleic acids containing any combination of natural or modified RNAand/or DNA, including, but not limited to such nucleic acids havingmodified nucleobases. By way of further example and without limitation,an oligomeric compound having the nucleobase sequence “ATCGATCG”encompasses any oligomeric compounds having such nucleobase sequence,whether modified or unmodified, including, but not limited to, suchcompounds comprising RNA bases, such as those having sequence “AUCGAUCG”and those having some DNA bases and some RNA bases such as “AUCGATCG”and oligomeric compounds having other modified bases, such as“AT^(me)CGAUCG,” wherein ^(me)C indicates a cytosine base comprising amethyl group at the 5-position.

Nucleic acids described herein by Isis Number (Isis NO.) comprise acombination of nucleobase sequence and certain identified modifications.

Certain Modified Oligonucleotides

In certain embodiments, a modified oligonucleotide consists of 15 to 30linked nucleosides. In certain embodiments, a modified oligonucleotideconsists of 19 to 24 linked nucleosides. In certain embodiments, amodified oligonucleotide consists of 21 to 24 linked nucleosides. Incertain embodiments, a modified oligonucleotide consists of 15 linkednucleosides. In certain embodiments, a modified oligonucleotide consistsof 16 linked nucleosides. In certain embodiments, a modifiedoligonucleotide consists of 17 linked nucleosides. In certainembodiments, a modified oligonucleotide consists of 18 linkednucleosides. In certain embodiments, a modified oligonucleotide consistsof 19 linked nucleosides. In certain embodiments, a modifiedoligonucleotide consists of 20 linked nucleosides. In certainembodiments, a modified oligonucleotide consists of 21 linkednucleosides. In certain embodiments, a modified oligonucleotide consistsof 22 linked nucleosides. In certain embodiments, a modifiedoligonucleotide consists of 23 linked nucleosides. In certainembodiments, a modified oligonucleotide consists of 24 linkednucleosides. In certain embodiments, a modified oligonucleotide consistsof 25 linked nucleosides. In certain embodiments, a modifiedoligonucleotide consists of 26 linked nucleosides. In certainembodiments, a modified oligonucleotide consists of 27 linkednucleosides. In certain embodiments, a modified oligonucleotide consistsof 28 linked nucleosides. In certain embodiments, a modifiedoligonucleotide consists of 29 linked nucleosides. In certainembodiments, a modified oligonucleotide consists of 30 linkednucleosides.

Certain Modifications

Modified oligonucleotides of the present invention comprise one or moremodifications to a nucleobase, sugar, and/or internucleoside linkage. Amodified nucleobase, sugar, and/or internucleoside linkage may beselected over an unmodified form because of desirable properties suchas, for example, enhanced cellular uptake, enhanced affinity for otheroligonucleotides or nucleic acid targets and increased stability in thepresence of nucleases.

In certain embodiments, a modified oligonucleotide of the presentinvention comprises one or more modified nucleosides. In certain suchembodiments, a modified nucleoside is a stabilizing nucleoside. Anexample of a stabilizing nucleoside is a sugar-modified nucleoside.

In certain embodiments, a modified nucleoside is a sugar-modifiednucleoside. In certain such embodiments, the sugar-modified nucleosidescan further comprise a natural or modified heterocyclic base moietyand/or a natural or modified internucleoside linkage and may includefurther modifications independent from the sugar modification. Incertain embodiments, a sugar modified nucleoside is a 2′-modifiednucleoside, wherein the sugar ring is modified at the 2′ carbon fromnatural ribose or 2′-deoxy-ribose.

In certain embodiments, a 2′-modified nucleoside has a bicyclic sugarmoiety. In certain such embodiments, the bicyclic sugar moiety is a Dsugar in the alpha configuration. In certain such embodiments, thebicyclic sugar moiety is a D sugar in the beta configuration. In certainsuch embodiments, the bicyclic sugar moiety is an L sugar in the alphaconfiguration. In certain such embodiments, the bicyclic sugar moiety isan L sugar in the beta configuration.

In certain embodiments, the bicyclic sugar moiety comprises a bridgegroup between the 2′ and the 4′-carbon atoms. In certain suchembodiments, the bridge group comprises from 1 to 8 linked biradicalgroups. In certain embodiments, the bicyclic sugar moiety comprises from1 to 4 linked biradical groups. In certain embodiments, the bicyclicsugar moiety comprises 2 or 3 linked biradical groups. In certainembodiments, the bicyclic sugar moiety comprises 2 linked biradicalgroups. In certain embodiments, a linked biradical group is selectedfrom —O—, —S—, —N(R₁)—, —C(R₁)(R₂)—, —C(R₁)═C(R₁)—, —C(R₁)═N—,—C(═NR₁)—, —Si(R₁)(R₂)—, —S(═O)₂—, —S(═O)—, —C(═O)— and —C(═S)—; whereeach R₁ and R₂ is, independently, H, hydroxyl, C₁-C₁₂ alkyl, substitutedC₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀aryl, a heterocycle radical, a substituted heterocycle radical,heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical, substitutedC₅-C₇ alicyclic radical, halogen, substituted oxy (—O—), amino,substituted amino, azido, carboxyl, substituted carboxyl, acyl,substituted acyl, CN, thiol, substituted thiol, sulfonyl (S(═O)₂—H),substituted sulfonyl, sulfoxyl (S(═O)—H) or substituted sulfoxyl; andeach substituent group is, independently, halogen, C₁-C₁₂ alkyl,substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl,C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, amino, substituted amino,acyl, substituted acyl, C₁-C₁₂ aminoalkyl, C₁-C₁₂ aminoalkoxy,substituted C₁-C₁₂ aminoalkyl, substituted C₁-C₁₂ aminoalkoxy or aprotecting group.

In some embodiments, the bicyclic sugar moiety is bridged between the 2′and 4′ carbon atoms with a biradical group selected from —O—(CH₂)_(p)—,—O—CH₂—, —O—CH₂CH₂—, —O—CH(alkyl)-, —NH—(CH₂)_(p)—,—N(alkyl)-(CH₂)_(p)—, —O—CH(alkyl)-, —(CH(alkyl))-(CH₂)_(p)—,—NH—O—(CH₂)_(p)—, —N(alkyl)-O—(CH₂)_(p)—, or —O—N(alkyl)-(CH₂)_(p)—,wherein p is 1, 2, 3, 4 or 5 and each alkyl group can be furthersubstituted. In certain embodiments, p is 1, 2 or 3.

In certain embodiments, a 2′-modified nucleoside comprises a2′-substituent group selected from halo, allyl, amino, azido, SH, CN,OCN, CF₃, OCF₃, O—, S—, or N(R_(m))-alkyl; O—, S—, or N(R_(m))-alkenyl;O—, S— or N(R_(m))-alkynyl; O-alkylenyl-O-alkyl, alkynyl, alkaryl,aralkyl, O-alkaryl, O-aralkyl, O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(R_(m))(R_(n))or O—CH₂—C(═O)—N(R_(m))(R_(n)), where each R_(m) and R_(n) is,independently, H, an amino protecting group or substituted orunsubstituted C₁-C₁₀ alkyl. These 2′-substituent groups can be furthersubstituted with one or more substituent groups independently selectedfrom hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO₂),thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.

In certain embodiments, a 2′-modified nucleoside comprises a2′-substituent group selected from F, NH₂, N₃, OCF₃, O—CH₃, O(CH₂)₃NH₂,CH₂—CH═CH₂, O—CH₂—CH═CH₂, OCH₂CH₂OCH₃, O(CH₂)₂SCH₃,O—(CH₂)₂—O—N(R_(m))(R_(n)), —O(CH₂)₂O(CH₂)₂N(CH₃)₂, and N-substitutedacetamide (O—CH₂—C(═O)—N(R_(m))(R_(n)) where each R_(m) and R_(n) is,independently, H, an amino protecting group or substituted orunsubstituted C₁-C₁₀ alkyl.

In certain embodiments, a 2′-modified nucleoside comprises a2′-substituent group selected from F, OCF₃, O—CH₃, OCH₂CH₂OCH₃,2′-O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(CH₃)₂, —O(CH₂)₂O(CH₂)₂N—(CH₃)₂, andO—CH₂—C(═O)—N(H)CH₃.

In certain embodiments, a 2′-modified nucleoside comprises a2′-substituent group selected from F, O—CH₃, and OCH₂CH₂OCH₃.

In certain embodiments, a sugar-modified nucleoside is a 4′-thiomodified nucleoside. In certain embodiments, a sugar-modified nucleosideis a 4′-thio-2′-modified nucleoside. A 4′-thio modified nucleoside has aβ-D-ribonucleoside where the 4′-0 replaced with 4′-S. A4′-thio-2′-modified nucleoside is a 4′-thio modified nucleoside havingthe 2′-OH replaced with a 2′-substituent group. Suitable 2′-substituentgroups include 2′-OCH₃, 2′-O—(CH₂)₂—OCH₃, and 2′-F.

In certain embodiments, a modified oligonucleotide of the presentinvention comprises one or more internucleoside modifications. Incertain such embodiments, each internucleoside linkage of a modifiedoligonucleotide is a modified internucleoside linkage. In certainembodiments, a modified internucleoside linkage comprises a phosphorusatom.

In certain embodiments, a modified oligonucleotide of the presentinvention comprises at least one phosphorothioate internucleosidelinkage. In certain embodiments, each internucleoside linkage of amodified oligonucleotide is a phosphorothioate internucleoside linkage.

In certain embodiments, a modified internucleoside linkage does notcomprise a phosphorus atom. In certain such embodiments, aninternucleoside linkage is formed by a short chain alkyl internucleosidelinkage. In certain such embodiments, an internucleoside linkage isformed by a cycloalkyl internucleoside linkages. In certain suchembodiments, an internucleoside linkage is formed by a mixed heteroatomand alkyl internucleoside linkage. In certain such embodiments, aninternucleoside linkage is formed by a mixed heteroatom and cycloalkylinternucleoside linkages. In certain such embodiments, aninternucleoside linkage is formed by one or more short chainheteroatomic internucleoside linkages. In certain such embodiments, aninternucleoside linkage is formed by one or more heterocyclicinternucleoside linkages. In certain such embodiments, aninternucleoside linkage has an amide backbone. In certain suchembodiments, an internucleoside linkage has mixed N, O, S and CH₂component parts.

In certain embodiments, a modified oligonucleotide comprises one or moremodified nucleobases. In certain embodiments, a modified oligonucleotidecomprises one or more 5-methylcytosines. In certain embodiments, eachcytosine of a modified oligonucleotide comprises a 5-methylcytosine.

In certain embodiments, a modified nucleobase is selected from5-hydroxymethyl cytosine, 7-deazaguanine and 7-deazaadenine. In certainembodiments, a modified nucleobase is selected from 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. In certainembodiments, a modified nucleobase is selected from 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines,including 2 aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.

In certain embodiments, a modified nucleobase comprises a polycyclicheterocycle. In certain embodiments, a modified nucleobase comprises atricyclic heterocycle. In certain embodiments, a modified nucleobasecomprises a phenoxazine derivative. In certain embodiments, thephenoxazine can be further modified to form a nucleobase known in theart as a G-clamp.

Certain Oligonucleotide Motifs

Suitable motifs for modified oligonucleotides of the present inventioninclude, but are not limited to, fully modified, uniformly modified,positionally modified, and gapmer. Modified oligonucleotides having afully modified motif, including a uniformly modified motif, may bedesigned to target mature miRNAs. Alternatively, modifiedoligonucleotides having a fully modified motif, including a uniformlymodified motif, may be designed to target certain sites of pri-miRNAs orpre-miRNAs, to block the processing of miRNA precursors into maturemiRNAs. Modified oligonucleotides having a fully modified motif oruniformly modified motif are effective inhibitors of miRNA activity.

In certain embodiments, a fully modified oligonucleotide comprises asugar modification at each nucleoside. In certain such embodiments,pluralities of nucleosides are 2′-O-methoxyethyl nucleosides and theremaining nucleosides are 2′-fluoro nucleosides. In certain suchembodiments, each of a plurality of nucleosides is a 2′-O-methoxyethylnucleoside and each of a plurality of nucleosides is a bicyclicnucleoside. In certain such embodiments, a fully modifiedoligonucleotide further comprises at least one modified internucleosidelinkage. In certain such embodiments, each internucleoside linkage of afully sugar-modified oligonucleotide is a modified internucleosidelinkage. In certain embodiments, a fully sugar-modified oligonucleotidefurther comprises at least one phosphorothioate internucleoside linkage.In certain such embodiments, each internucleoside linkage of a fullysugar-modified oligonucleotide is a phosphorothioate internucleosidelinkage.

In certain embodiments, a fully modified oligonucleotide is modified ateach internucleoside linkage. In certain such embodiments, eachinternucleoside linkage of a fully modified oligonucleotide is aphosphorothioate internucleoside linkage.

In certain embodiments, a uniformly modified oligonucleotide comprisesthe same sugar modification at each nucleoside. In certain suchembodiments, each nucleoside of a modified oligonucleotide comprises a2′-O-methoxyethyl sugar modification. In certain embodiments, eachnucleoside of a modified oligonucleotide comprises a 2′-O-methyl sugarmodification. In certain embodiments, each nucleoside of a modifiedoligonucleotide comprises a 2′-fluoro sugar modification. In certainsuch embodiments, a uniformly modified oligonucleotide further comprisesat least one modified internucleoside linkage. In certain suchembodiments, each internucleoside linkage of a uniformly sugar-modifiedoligonucleotide is a modified internucleoside linkage. In certainembodiments, a uniformly sugar-modified oligonucleotide furthercomprises at least one phosphorothioate internucleoside linkage. Incertain such embodiments, each internucleoside linkage of a uniformlysugar-modified oligonucleotide is a phosphorothioate internucleosidelinkage.

In certain embodiments, a uniformly modified oligonucleoside comprisesthe same internucleoside linkage modifications throughout. In certainsuch embodiments, each internucleoside linkage of a uniformly modifiedoligonucleotide is a phosphorothioate internucleoside linkage.

Table 1 illustrates certain uniformly modified oligonucleotidescomplementary to the miRNAs described herein. Each nucleoside comprisesa 2′-O-methoxyethyl sugar, each internucleoside linkage isphosphorothioate, and each cytosine is a 5-methylcytosine.

TABLE 1 Modified SEQ ID Version 10 Oligonucleotide # NO miRNA Sanger mirID 327917 1 miR-21 hsa-miR-21 341787 2 miR-125a hsa-miR-125a-5p 341802 3mir-191 hsa-miR-191 401852 4 mir-210 hsa-miR-210 327920 5 mir-222hsa-miR-222 379242 6 miR-422b hsa-miR-378 379243 7 mir-423hsa-miR-423-3p 399329 8 miR-638 hsa-miR-638

In certain embodiments, a positionally modified oligonucleotidecomprises regions of linked nucleosides, where each nucleoside of eachregion comprises the same sugar moiety, and where each nucleoside ofeach region comprises a sugar moiety different from that of an adjacentregion.

In certain embodiments, a positionally modified oligonucleotidecomprises at least 10 2′-fluoro modified nucleosides. Such apositionally modified oligonucleotide may be represented by thefollowing formula I:5′-T₁-(Nu₁-L₁)_(n1)-(Nu₂-L₂)_(n2)-Nu₂-(L₃-Nu₃)_(n3)-T₂₋₃′, wherein:

-   -   each Nu₁ and Nu₃ is, independently, a stabilizing nucleoside;    -   at least 10 Nu₂ are 2′-fluoro nucleosides;    -   each L₁, L₂ and L₃ is, independently, an internucleoside        linkage;    -   each T₁ and T₂ is, independently, H, a hydroxyl protecting        group, an optionally linked conjugate group or a capping group;

n₁ is from 0 to about 3;

n₂ is from about 14 to about 22;

n₃ is from 0 to about 3; and

provided that dm is 0 then T₁ is not H or a hydroxyl protecting group,and if n₃ is 0, then T₂ is not H or a hydroxyl protecting group.

In certain such embodiments, n₁ and n₃ are each, independently, from 1to about 3. In certain embodiments, n₁ and n₃ are each, independently,from 2 to about 3. In certain embodiments, n₁ is 1 or 2 and n₃ is 2 or3. In certain embodiments, n₁ and n₃ are each 2. In certain embodiments,at least one of n₁ and n₃ is greater than zero. In certain embodiments,n₁ and n₃ is each greater than zero. In certain embodiments, one of n₁and n₃ is greater than zero. In certain embodiments, one of n₁ and n₃ isgreater than one.

In certain embodiments, n₂ is from 16 to 20. In certain embodiments, n₂is from 17 to 19. In certain embodiments, n₂ is 18. In certainembodiments, n₂ is 19. In certain embodiments, n₂ is 20.

In certain embodiments, about 2 to about 8 of the Nu₂ nucleosides arestabilizing nucleosides. In certain embodiments, from about 2 to about 6of the Nu₂ nucleosides are stabilizing nucleosides. In certainembodiments, from about 3 to about 4 of the Nu₂ nucleosides arestabilizing nucleosides. In certain embodiments, 3 of the Nu₂nucleosides are stabilizing nucleosides.

In certain embodiments, each of the Nu₂ stabilizing nucleosides isseparated from the Nu₃ stabilizing nucleosides by from 2 to about 82′-fluoro nucleosides. In certain embodiments each of the Nu₂stabilizing nucleosides is separated from the Nu₃ stabilizingnucleosides by from 3 to about 8 2′-fluoro nucleosides. In certainembodiments each of the Nu₂ stabilizing nucleosides is separated fromthe Nu₃ stabilizing nucleosides by from 5 to about 8 2′-fluoronucleosides.

In certain embodiments, a modified oligonucleotide comprises from 2 toabout 6 Nu₂ stabilizing nucleosides. In certain embodiments, a modifiedoligonucleotide comprises 3 Nu₂ stabilizing nucleosides.

In certain embodiments, each of the Nu₂ stabilizing nucleosides islinked together in one contiguous sequence. In certain embodiments, atleast two of the Nu₂ stabilizing nucleosides are separated by at leastone of the 2′-fluoro nucleosides. In certain embodiments, each of theNu₂ stabilizing nucleosides is separated by at least one of the2′-fluoro nucleosides.

In certain embodiments, at least two contiguous sequences of the Nu₂2′-fluoro nucleosides are separated by at least one of the stabilizingnucleosides wherein each of the contiguous sequences have the samenumber of 2′-fluoro nucleosides.

In certain embodiments, T₁ and T₂ are each, independently, H or ahydroxyl protecting group. In certain embodiments, at least one of T₁and T₂ is 4,4′-dimethoxytrityl. In certain embodiments, at least one ofT₁ and T₂ is an optionally linked conjugate group. In certainembodiments, at least one of T₁ and T₂ is a capping group. In certainembodiments, the capping group is an inverted deoxy abasic group.

In certain embodiments, a positionally modified oligonucleotidecomprises at least one modified internucleoside linkage. In certain suchembodiments, each internucleoside linkage of a positionally modifiedoligonucleoside is a modified internucleoside linkage. In certainembodiments, at least one internucleoside linkage of a positionallymodified oligonucleotide is a phosphorothioate internucleoside linkage.In certain such embodiments, each internucleoside linkage of apositionally modified oligonucleotide is a phosphorothioateinternucleoside linkage.

In certain embodiments, a positionally modified motif is represented bythe following formula II, which represents a modified oligonucleotideconsisting of linked nucleosides:T₁-(Nu₁)_(n1)-(Nu₂)_(n2)-(Nu₃)_(n3)(Nu₄)_(n4)-(Nu₅)_(n5)-T₂, wherein:

Nu₁ and Nu₅ are, independently, 2′ stabilizing nucleosides;

Nu₂ and Nu₄ are 2′-fluoro nucleosides;

Nu₃ is a 2′-modified nucleoside;

each of n₁ and n₅ is, independently, from 0 to 3;

the sum of n₂ plus n₄ is between 10 and 25;

n₃ is from 0 and 5; and

each T₁ and T₂ is, independently, H, a hydroxyl protecting group, anoptionally linked conjugate group or a capping group.

In certain embodiments, the sum of n₂ and n₄ is 16. In certainembodiments, the sum of n₂ and n₄ is 17. In certain embodiments, the sumof n₂ and n₄ is 18. In certain embodiments, n₁ is 2; n₃ is 2 or 3; andn₅ is 2.

In certain embodiments, Nu₁ and Nu₅ are, independently, 2′-modifiednucleosides.

In certain embodiments, Nu₁ is O—(CH₂)₂—OCH₃, Nu₃ is O—(CH₂)₂—OCH₃,Nu₅O—(CH₂)₂—OCH₃, T₁ is H and T₂ is H.

In certain embodiments, a modified oligonucleotide complementary to amiRNA and consisting of 21 linked nucleosides has a Formula II selectedfrom Table 2, where each internucleoside linkage is a phosphorothioateinternucleoside linkage. In certain embodiments, a modifiedoligonucleotide having a Formula II selected from Table 2 has anucleobase sequence selected from the nucleobase sequences recited inSEQ ID NOs 24 and 27.

TABLE 2  n₁ n₂ n₃ n₄ n₅ Nu₁ Nu₃ Nu₅ T₁ T₂ 2 17 0 0 2 2′-MOE 2′-MOE2′-MOE H H 2 2 2 13 2 2′-MOE 2′-MOE 2′-MOE H H 2 3 2 12 2 2′-MOE 2′-MOE2′-MOE H H 2 4 2 11 2 2′-MOE 2′-MOE 2′-MOE H H 2 5 2 10 2 2′-MOE 2′-MOE2′-MOE H H 2 6 2 9 2 2′-MOE 2′-MOE 2′-MOE H H 2 7 2 8 2 2′-MOE 2′-MOE2′-MOE H H 2 8 2 7 2 2′-MOE 2′-MOE 2′-MOE H H 2 9 2 6 2 2′-MOE 2′-MOE2′-MOE H H 2 10 2 5 2 2′-MOE 2′-MOE 2′-MOE H H 2 11 2 4 2 2′-MOE 2′-MOE2′-MOE H H 2 12 2 3 2 2′-MOE 2′-MOE 2′-MOE H H 2 13 2 2 2 2′-MOE 2′-MOE2′-MOE H H 2 2 3 12 2 2′-MOE 2′-MOE 2′-MOE H H 2 3 3 11 2 2′-MOE 2′-MOE2′-MOE H H 2 4 3 10 2 2′-MOE 2′-MOE 2′-MOE H H 2 5 3 9 2 2′-MOE 2′-MOE2′-MOE H H 2 6 3 8 2 2′-MOE 2′-MOE 2′-MOE H H 2 7 3 7 2 2′-MOE 2′-MOE2′-MOE H H 2 8 3 6 2 2′-MOE 2′-MOE 2′-MOE H H 2 9 3 5 2 2′-MOE 2′-MOE2′-MOE H H 2 10 3 4 2 2′-MOE 2′-MOE 2′-MOE H H 2 11 3 3 2 2′-MOE 2′-MOE2′-MOE H H 2 12 3 2 2 2′-MOE 2′-MOE 2′-MOE H H 2 8 6 3 2 2′-MOE 2′-MOE2′-MOE H H

In certain embodiments, a modified oligonucleotide complementary to amiRNA and consisting of 22 linked nucleosides has a Formula II selectedfrom Table 3, where each internucleoside linkage is a phosphorothioateinternucleoside linkage. In certain embodiments, a modifiedoligonucleotide having a Formula II selected from Table 3 has anucleobase sequence selected from the nucleobase sequences recited inSEQ ID NOs 17, 20, 22, 26, and 28.

TABLE 3  n₁ n₂ n₃ n₄ n₅ Nu₁ Nu₃ Nu₅ T₁ T₂ 2 18 0 0 2 2′-MOE 2′-MOE2′-MOE H H 2 2 2 14 2 2′-MOE 2′-MOE 2′-MOE H H 2 3 2 13 2 2′-MOE 2′-MOE2′-MOE H H 2 4 2 12 2 2′-MOE 2′-MOE 2′-MOE H H 2 5 2 11 2 2′-MOE 2′-MOE2′-MOE H H 2 6 2 10 2 2′-MOE 2′-MOE 2′-MOE H H 2 7 2 9 2 2′-MOE 2′-MOE2′-MOE H H 2 8 2 8 2 2′-MOE 2′-MOE 2′-MOE H H 2 9 2 7 2 2′-MOE 2′-MOE2′-MOE H H 2 10 2 6 2 2′-MOE 2′-MOE 2′-MOE H H 2 11 2 5 2 2′-MOE 2′-MOE2′-MOE H H 2 12 2 4 2 2′-MOE 2′-MOE 2′-MOE H H 2 13 2 3 2 2′-MOE 2′-MOE2′-MOE H H 2 14 2 2 2 2′-MOE 2′-MOE 2′-MOE H H 2 2 3 13 2 2′-MOE 2′-MOE2′-MOE H H 2 3 3 12 2 2′-MOE 2′-MOE 2′-MOE H H 2 4 3 11 2 2′-MOE 2′-MOE2′-MOE H H 2 5 3 10 2 2′-MOE 2′-MOE 2′-MOE H H 2 6 3 9 2 2′-MOE 2′-MOE2′-MOE H H 2 7 3 8 2 2′-MOE 2′-MOE 2′-MOE H H 2 8 3 7 2 2′-MOE 2′-MOE2′-MOE H H 2 9 3 6 2 2′-MOE 2′-MOE 2′-MOE H H 2 10 3 5 2 2′-MOE 2′-MOE2′-MOE H H 2 11 3 4 2 2′-MOE 2′-MOE 2′-MOE H H 2 12 3 3 2 2′-MOE 2′-MOE2′-MOE H H 2 13 3 2 2 2′-MOE 2′-MOE 2′-MOE H H 2 8 6 4 2 2′-MOE 2′-MOE2′-MOE H H

In certain embodiments, a modified oligonucleotide complementary to amiRNA and consisting of 23 linked nucleosides has a Formula II selectedfrom Table 4, where each internucleoside linkage is a phosphorothioateinternucleoside linkage. In certain embodiments, a modifiedoligonucleotide having a Formula II selected from Table 4 has anucleobase sequence selected from the nucleobase sequences recited inSEQ ID NOs 18, 21, and 23.

TABLE 4 n₁ n₂ n₃ n₄ n₅ Nu₁ Nu₃ Nu₅ T₁ T₂ 2 19 0 0 2 2′-MOE 2′-MOE 2′-MOEH H 2 2 2 15 2 2′-MOE 2′-MOE 2′-MOE H H 2 3 2 14 2 2′-MOE 2′-MOE 2′-MOEH H 2 4 2 13 2 2′-MOE 2′-MOE 2′-MOE H H 2 5 2 12 2 2′-MOE 2′-MOE 2′-MOEH H 2 6 2 11 2 2′-MOE 2′-MOE 2′-MOE H H 2 7 2 10 2 2′-MOE 2′-MOE 2′-MOEH H 2 8 2 9 2 2′-MOE 2′-MOE 2′-MOE H H 2 9 2 8 2 2′-MOE 2′-MOE 2′-MOE HH 2 10 2 7 2 2′-MOE 2′-MOE 2′-MOE H H 2 11 2 6 2 2′-MOE 2′-MOE 2′-MOE HH 2 12 2 5 2 2′-MOE 2′-MOE 2′-MOE H H 2 13 2 4 2 2′-MOE 2′-MOE 2′-MOE HH 2 14 2 3 2 2′-MOE 2′-MOE 2′-MOE H H 2 15 2 2 2 2′-MOE 2′-MOE 2′-MOE HH 2 2 3 14 2 2′-MOE 2′-MOE 2′-MOE H H 2 3 3 13 2 2′-MOE 2′-MOE 2′-MOE HH 2 4 3 12 2 2′-MOE 2′-MOE 2′-MOE H H 2 5 3 11 2 2′-MOE 2′-MOE 2′-MOE HH 2 6 3 10 2 2′-MOE 2′-MOE 2′-MOE H H 2 7 3 9 2 2′-MOE 2′-MOE 2′-MOE H H2 8 3 8 2 2′-MOE 2′-MOE 2′-MOE H H 2 9 3 7 2 2′-MOE 2′-MOE 2′-MOE H H 210 3 6 2 2′-MOE 2′-MOE 2′-MOE H H 2 11 3 5 2 2′-MOE 2′-MOE 2′-MOE H H 212 3 4 2 2′-MOE 2′-MOE 2′-MOE H H 2 13 3 3 2 2′-MOE 2′-MOE 2′-MOE H H 214 3 2 2 2′-MOE 2′-MOE 2′-MOE H H 2 8 6 5 2 2′-MOE 2′-MOE 2′-MOE H H

In certain embodiments, a modified oligonucleotide complementary to amiRNA and consisting of 24 linked nucleosides has a Formula II selectedfrom Table 5, where each internucleoside linkage is a phosphorothioateinternucleoside linkage. In certain embodiments, a modifiedoligonucleotide having a Formula II selected from Table 5 has anucleobase sequence selected from the nucleobase sequences recited inSEQ ID NOs 19 and 23.

TABLE 5 n₁ n₂ n₃ n₄ n₅ Nu₁ Nu₃ Nu₅ T₁ T₂ 2 20 0 0 2 2′-MOE 2′-MOE 2′-MOEH H 2 2 2 16 2 2′-MOE 2′-MOE 2′-MOE H H 2 3 2 15 2 2′-MOE 2′-MOE 2′-MOEH H 2 4 2 14 2 2′-MOE 2′-MOE 2′-MOE H H 2 5 2 13 2 2′-MOE 2′-MOE 2′-MOEH H 2 6 2 12 2 2′-MOE 2′-MOE 2′-MOE H H 2 7 2 11 2 2′-MOE 2′-MOE 2′-MOEH H 2 8 2 10 2 2′-MOE 2′-MOE 2′-MOE H H 2 9 2 9 2 2′-MOE 2′-MOE 2′-MOE HH 2 10 2 8 2 2′-MOE 2′-MOE 2′-MOE H H 2 11 2 7 2 2′-MOE 2′-MOE 2′-MOE HH 2 12 2 6 2 2′-MOE 2′-MOE 2′-MOE H H 2 13 2 5 2 2′-MOE 2′-MOE 2′-MOE HH 2 14 2 4 2 2′-MOE 2′-MOE 2′-MOE H H 2 15 2 3 2 2′-MOE 2′-MOE 2′-MOE HH 2 16 2 2 2 2′-MOE 2′-MOE 2′-MOE H H 2 2 3 15 2 2′-MOE 2′-MOE 2′-MOE HH 2 3 3 14 2 2′-MOE 2′-MOE 2′-MOE H H 2 4 3 13 2 2′-MOE 2′-MOE 2′-MOE HH 2 5 3 12 2 2′-MOE 2′-MOE 2′-MOE H H 2 6 3 11 2 2′-MOE 2′-MOE 2′-MOE HH 2 7 3 10 2 2′-MOE 2′-MOE 2′-MOE H H 2 8 3 9 2 2′-MOE 2′-MOE 2′-MOE H H2 9 3 8 2 2′-MOE 2′-MOE 2′-MOE H H 2 10 3 7 2 2′-MOE 2′-MOE 2′-MOE H H 211 3 6 2 2′-MOE 2′-MOE 2′-MOE H H 2 12 3 5 2 2′-MOE 2′-MOE 2′-MOE H H 213 3 4 2 2′-MOE 2′-MOE 2′-MOE H H 2 14 3 3 2 2′-MOE 2′-MOE 2′-MOE H H 215 3 2 2 2′-MOE 2′-MOE 2′-MOE H H 2 8 6 6 2 2′-MOE 2′-MOE 2′-MOE H H

In certain embodiments, a modified oligonucleotide complementary to amiRNA and consisting of 25 linked nucleosides has a Formula II selectedfrom Table 6, where each internucleoside linkage is a phosphorothioateinternucleoside linkage. In certain embodiments, a modifiedoligonucleotide having a Formula II selected from Table 6 has thenucleobase sequence recited in SEQ ID NOs 30.

TABLE 6 n₁ n₂ n₃ n₄ n₅ Nu₁ Nu₃ Nu₅ T₁ T₂ 2 21 0 0 2 2′-MOE 2′-MOE 2′-MOEH H 2 2 2 17 2 2′-MOE 2′-MOE 2′-MOE H H 2 3 2 16 2 2′-MOE 2′-MOE 2′-MOEH H 2 4 2 15 2 2′-MOE 2′-MOE 2′-MOE H H 2 5 2 14 2 2′-MOE 2′-MOE 2′-MOEH H 2 6 2 13 2 2′-MOE 2′-MOE 2′-MOE H H 2 7 2 12 2 2′-MOE 2′-MOE 2′-MOEH H 2 8 2 11 2 2′-MOE 2′-MOE 2′-MOE H H 2 9 2 10 2 2′-MOE 2′-MOE 2′-MOEH H 2 10 2 9 2 2′-MOE 2′-MOE 2′-MOE H H 2 11 2 8 2 2′-MOE 2′-MOE 2′-MOEH H 2 12 2 7 2 2′-MOE 2′-MOE 2′-MOE H H 2 13 2 6 2 2′-MOE 2′-MOE 2′-MOEH H 2 14 2 5 2 2′-MOE 2′-MOE 2′-MOE H H 2 15 2 4 2 2′-MOE 2′-MOE 2′-MOEH H 2 16 2 3 2 2′-MOE 2′-MOE 2′-MOE H H 2 17 2 2 2 2′-MOE 2′-MOE 2′-MOEH H 2 2 3 16 2 2′-MOE 2′-MOE 2′-MOE H H 2 3 3 15 2 2′-MOE 2′-MOE 2′-MOEH H 2 4 3 14 2 2′-MOE 2′-MOE 2′-MOE H H 2 5 3 13 2 2′-MOE 2′-MOE 2′-MOEH H 2 6 3 12 2 2′-MOE 2′-MOE 2′-MOE H H 2 7 3 11 2 2′-MOE 2′-MOE 2′-MOEH H 2 8 3 10 2 2′-MOE 2′-MOE 2′-MOE H H 2 9 3 9 2 2′-MOE 2′-MOE 2′-MOE HH 2 10 3 8 2 2′-MOE 2′-MOE 2′-MOE H H 2 11 3 7 2 2′-MOE 2′-MOE 2′-MOE HH 2 12 3 6 2 2′-MOE 2′-MOE 2′-MOE H H 2 13 3 5 2 2′-MOE 2′-MOE 2′-MOE HH 2 14 3 4 2 2′-MOE 2′-MOE 2′-MOE H H 2 15 3 3 2 2′-MOE 2′-MOE 2′-MOE HH 2 16 3 2 2 2′-MOE 2′-MOE 2′-MOE H H 2 8 6 7 2 2′-MOE 2′-MOE 2′-MOE H H

A modified oligonucleotide having a gapmer motif may have an internalregion consisting of linked 2′-deoxynucleotides, and external regionsconsisting of linked 2′-modified nucleosides. Such a gapmer may bedesigned to elicit RNase H cleavage of a miRNA precursor. The internal2′-deoxynucleoside region serves as a substrate for RNase H, allowingthe cleavage of the miRNA precursor to which a modified oligonucleotideis targeted. In certain embodiments, each nucleoside of each externalregion comprises the same 2′-modified nucleoside. In certainembodiments, one external region is uniformly comprised of a first2′-modified nucleoside and the other external region is uniformlycomprised of a second 2′-modified nucleoside.

A modified oligonucleotide having a gapmer motif may have a sugarmodification at each nucleoside. In certain embodiments, the internalregion is uniformly comprised of a first 2′-modified nucleoside and eachof the wings is uniformly comprised of a second 2′-modified nucleoside.In certain such embodiments, the internal region is uniformly comprisedof 2′-fluoro nucleosides and each external region is uniformly comprisedof 2′-O-methoxyethyl nucleosides.

In certain embodiments, each external region of a gapmer consists oflinked 2′-O-methoxyethyl nucleosides. In certain embodiments, eachexternal region of a gapmer consists of linked 2′-O-methyl nucleosides.In certain embodiments, each external region of a gapmer consists of2′-fluoro nucleosides. In certain embodiments, each external region of agapmer consists of linked bicyclic nucleosides.

In certain embodiments, each nucleoside of one external region of agapmer comprises 2′-O-methoxyethyl nucleosides and each nucleoside ofthe other external region comprises a different 2′-modification. Incertain such embodiments, each nucleoside of one external region of agapmer comprises 2′-O-methoxyethyl nucleosides and each nucleoside ofthe other external region comprises 2′-O-methyl nucleosides. In certainsuch embodiments, each nucleoside of one external region of a gapmercomprises 2′-O-methoxyethyl nucleosides and each nucleoside of the otherexternal region comprises 2′-fluoro nucleosides. In certain suchembodiments, each nucleoside of one external region of a gapmercomprises 2′-O-methyl nucleosides and each nucleoside of the otherexternal region comprises 2′-fluoro nucleosides. In certain suchembodiments, each nucleoside of one external region of a gapmercomprises 2′-O-methoxyethyl nucleosides and each nucleoside of the otherexternal region comprises bicyclic nucleosides. In certain suchembodiments, each nucleoside of one external region of a gapmercomprises 2′-O-methyl nucleosides and each nucleoside of the otherexternal region comprises bicyclic nucleosides.

In certain embodiments, nucleosides of one external region comprise twoor more sugar modifications. In certain embodiments, nucleosides of eachexternal region comprise two or more sugar modifications. In certainembodiments, at least one nucleoside of an external region comprises a2′-O-methoxyethyl sugar and at least one nucleoside of the same externalregion comprises a 2′-fluoro sugar. In certain embodiments, at least onenucleoside of an external region comprises a 2′-O-methoxyethyl sugar andat least one nucleoside of the same external region comprises a bicyclicsugar moiety. In certain embodiments, at least one nucleoside of anexternal region comprises a 2′-O-methyl sugar and at least onenucleoside of the same external region comprises a bicyclic sugarmoiety. In certain embodiments at least one nucleoside of an externalregion comprises a 2′-O-methyl sugar and at least one nucleoside of thesame external region comprises a 2′-fluoro sugar. In certainembodiments, at least one nucleoside of an external region comprises a2′-fluoro sugar and at least one nucleoside of the same external regioncomprises a bicyclic sugar moiety.

In certain embodiments, each external region of a gapmer consists of thesame number of linked nucleosides. In certain embodiments, one externalregion of a gapmer consists a number of linked nucleosides differentthat that of the other external region.

In certain embodiments, the external regions comprise, independently,from 1 to 6 nucleosides. In certain embodiments, an external regioncomprises 1 nucleoside. In certain embodiments, an external regioncomprises 2 nucleosides. In certain embodiments, an external regioncomprises 3 nucleosides. In certain embodiments, an external regioncomprises 4 nucleosides. In certain embodiments, an external regioncomprises 5 nucleosides. In certain embodiments, an external regioncomprises 6 nucleosides. In certain embodiments, the internal regionconsists of 17 to 28 linked nucleosides. In certain embodiments, aninternal region consists of 17 to 21 linked nucleosides. In certainembodiments, an internal region consists of 17 linked nucleosides. Incertain embodiments, an internal region consists of 18 linkednucleosides. In certain embodiments, an internal region consists of 19linked nucleosides. In certain embodiments, an internal region consistsof 20 linked nucleosides. In certain embodiments, an internal regionconsists of 21 linked nucleosides. In certain embodiments, an internalregion consists of 22 linked nucleosides. In certain embodiments, aninternal region consists of 23 linked nucleosides. In certainembodiments, an internal region consists of 24 linked nucleosides. Incertain embodiments, an internal region consists of 25 linkednucleosides. In certain embodiments, an internal region consists of 26linked nucleosides. In certain embodiments, an internal region consistsof 27 linked nucleosides. In certain embodiments, an internal regionconsists of 28 linked nucleosides.

Certain Additional Therapies

Cancer treatments often comprise more than one therapy. As such, incertain embodiments the present invention provides methods for treatingliver cancer comprising administering to a subject in need thereof acompound comprising a modified oligonucleotide complementary to a miRNA,or a precursor thereof, and further comprising administering at leastone additional therapy.

In certain embodiments, an additional therapy may also be designed totreat liver cancer, such as HCC. An additional therapy may be achemotherapeutic agent. Suitable chemotherapeutic agents include5-fluorouracil, gemcitabine, doxorubicine, mitomycin c, sorafenib,etoposide, carboplatin, epirubicin, irinotecan and oxaliplatin. Anadditional suitable chemotherapeutic agent includes a modifiedoligonucleotide, other than a modified oligonucleotide of the presentinvention, that is used to treat cancer. An additional therapy may besurgical resection of a liver tumor(s), liver transplantation, orchemoembolization.

In certain embodiments, an additional therapy may be designed to treat adisease other than liver cancer, including HCC. In certain suchembodiments, an additional therapy may be a treatment for hepatitis Cinfection or hepatitis B infection.

In certain embodiments, an additional therapy is a treatment forhepatitis C infection. Therapeutic agents for treatment of hepatitis Cinfection include interferons, for example, interferon alfa-2b,interferon alfa-2a, and interferon alfacon-1. Less frequent interferondosing can be achieved using pegylated interferon (interferon attachedto a polyethylene glycol moiety which significantly improves itspharmacokinetic profile). Combination therapy with interferon alfa-2b(pegylated and unpegylated) and ribavarin has also been shown to beefficacious for some patient populations. Other agents currently beingdeveloped include RNA replication inhibitors (e.g., ViroPharma's VP50406series), antisense agents (for example, anti-miR-122), therapeuticvaccines, protease inhibitors, helicase inhibitors and antibody therapy(monoclonal and polyclonal).

In certain embodiments, an additional therapy may be a pharmaceuticalagent that enhances the body's immune system, including low-dosecyclophosphamide, thymostimulin, vitamins and nutritional supplements(e.g., antioxidants, including vitamins A, C, E, beta-carotene, zinc,selenium, glutathione, coenzyme Q-10 and echinacea), and vaccines, e.g.,the immunostimulating complex (ISCOM), which comprises a vaccineformulation that combines a multimeric presentation of antigen and anadjuvant.

In certain such embodiments, the additional therapy is selected to treator ameliorate a side effect of one or more pharmaceutical compositionsof the present invention. Such side effects include, without limitation,injection site reactions, liver function test abnormalities, renalfunction abnormalities, liver toxicity, renal toxicity, central nervoussystem abnormalities, and myopathies. For example, increasedaminotransferase levels in serum may indicate liver toxicity or liverfunction abnormality. For example, increased bilirubin may indicateliver toxicity or liver function abnormality.

In certain embodiments, one or more pharmaceutical compositions of thepresent invention and one or more other pharmaceutical agents areadministered at the same time. In certain embodiments, one or morepharmaceutical compositions of the present invention and one or moreother pharmaceutical agents are administered at different times. Incertain embodiments, one or more pharmaceutical compositions of thepresent invention and one or more other pharmaceutical agents areprepared together in a single formulation. In certain embodiments, oneor more pharmaceutical compositions of the present invention and one ormore other pharmaceutical agents are prepared separately.

Certain Pharmaceutical Compositions

In certain embodiments, a compound comprising a modified oligonucleotidecomplementary to a miRNA, or precursor thereof, described herein isprepared as a pharmaceutical composition for the treatment of livercancer, including HCC. Suitable administration routes include, but arenot limited to, oral, rectal, transmucosal, intestinal, enteral,topical, suppository, through inhalation, intrathecal, intraventricular,intraperitoneal, intranasal, intraocular, intratumoral, and parenteral(e.g., intravenous, intramuscular, intramedullary, and subcutaneous). Anadditional suitable administration route includes chemoembolization. Incertain embodiments, pharmaceutical intrathecals are administered toachieve local rather than systemic exposures. For example,pharmaceutical compositions may be injected directly in the area ofdesired effect (e.g., into a tumor).

In certain embodiments, a pharmaceutical composition of the presentinvention is administered in the form of a dosage unit (e.g., tablet,capsule, bolus, etc.). In certain embodiments, such pharmaceuticalcompositions comprise a modified oligonucleotide in a dose selected from25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 110 mg, 115 mg, 120 mg,125 mg, 130 mg, 135 mg, 140 mg, 145 mg, 150 mg, 155 mg, 160 mg, 165 mg,170 mg, 175 mg, 180 mg, 185 mg, 190 mg, 195 mg, 200 mg, 205 mg, 210 mg,215 mg, 220 mg, 225 mg, 230 mg, 235 mg, 240 mg, 245 mg, 250 mg, 255 mg,260 mg, 265 mg, 270 mg, 270 mg, 280 mg, 285 mg, 290 mg, 295 mg, 300 mg,305 mg, 310 mg, 315 mg, 320 mg, 325 mg, 330 mg, 335 mg, 340 mg, 345 mg,350 mg, 355 mg, 360 mg, 365 mg, 370 mg, 375 mg, 380 mg, 385 mg, 390 mg,395 mg, 400 mg, 405 mg, 410 mg, 415 mg, 420 mg, 425 mg, 430 mg, 435 mg,440 mg, 445 mg, 450 mg, 455 mg, 460 mg, 465 mg, 470 mg, 475 mg, 480 mg,485 mg, 490 mg, 495 mg, 500 mg, 505 mg, 510 mg, 515 mg, 520 mg, 525 mg,530 mg, 535 mg, 540 mg, 545 mg, 550 mg, 555 mg, 560 mg, 565 mg, 570 mg,575 mg, 580 mg, 585 mg, 590 mg, 595 mg, 600 mg, 605 mg, 610 mg, 615 mg,620 mg, 625 mg, 630 mg, 635 mg, 640 mg, 645 mg, 650 mg, 655 mg, 660 mg,665 mg, 670 mg, 675 mg, 680 mg, 685 mg, 690 mg, 695 mg, 700 mg, 705 mg,710 mg, 715 mg, 720 mg, 725 mg, 730 mg, 735 mg, 740 mg, 745 mg, 750 mg,755 mg, 760 mg, 765 mg, 770 mg, 775 mg, 780 mg, 785 mg, 790 mg, 795 mg,and 800 mg. In certain such embodiments, a pharmaceutical composition ofthe present invention comprises a dose of modified oligonucleotideselected from 25 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300mg, 350 mg, 400 mg, 500 mg, 600 mg, 700 mg, and 800 mg.

In certain embodiments, a pharmaceutical agent is sterile lyophilizedmodified oligonucleotide that is reconstituted with a suitable diluent,e.g., sterile water for injection or sterile saline for injection. Thereconstituted product is administered as a subcutaneous injection or asan intravenous infusion after dilution into saline. The lyophilized drugproduct consists of a modified oligonucleotide which has been preparedin water for injection, or in saline for injection, adjusted to pH7.0-9.0 with acid or base during preparation, and then lyophilized. Thelyophilized modified oligonucleotide may be 25-800 mg of a modifiedoligonucleotide. It is understood that this encompasses 25, 50, 75, 100,125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 425, 450, 475,500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, and 800 mgof modified lyophilized oligonucleotide. The lyophilized drug productmay be packaged in a 2 mL Type I, clear glass vial (ammoniumsulfate-treated), stoppered with a bromobutyl rubber closure and sealedwith an aluminum FLIP-OFF® overseal.

In certain embodiments, the compositions of the present invention mayadditionally contain other adjunct components conventionally found inpharmaceutical compositions, at their art-established usage levels.Thus, for example, the compositions may contain additional, compatible,pharmaceutically-active materials such as, for example, antipruritics,astringents, local anesthetics or anti-inflammatory agents, or maycontain additional materials useful in physically formulating variousdosage forms of the compositions of the present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. However, such materials, when added, should notunduly interfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the oligonucleotide(s) of the formulation.

In certain embodiments, pharmaceutical compositions of the presentinvention comprise one or more modified oligonucleotides and one or moreexcipients. In certain such embodiments, excipients are selected fromwater, salt solutions, alcohol, polyethylene glycols, gelatin, lactose,amylase, magnesium stearate, talc, silicic acid, viscous paraffin,hydroxymethylcellulose and polyvinylpyrrolidone.

In certain embodiments, a pharmaceutical composition of the presentinvention is prepared using known techniques, including, but not limitedto mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or tabletting processes.

In certain embodiments, a pharmaceutical composition of the presentinvention is a liquid (e.g., a suspension, elixir and/or solution). Incertain of such embodiments, a liquid pharmaceutical composition isprepared using ingredients known in the art, including, but not limitedto, water, glycols, oils, alcohols, flavoring agents, preservatives, andcoloring agents.

In certain embodiments, a pharmaceutical composition of the presentinvention is a solid (e.g., a powder, tablet, and/or capsule). Incertain of such embodiments, a solid pharmaceutical compositioncomprising one or more oligonucleotides is prepared using ingredientsknown in the art, including, but not limited to, starches, sugars,diluents, granulating agents, lubricants, binders, and disintegratingagents.

In certain embodiments, a pharmaceutical composition of the presentinvention is formulated as a depot preparation. Certain such depotpreparations are typically longer acting than non-depot preparations. Incertain embodiments, such preparations are administered by implantation(for example subcutaneously or intramuscularly) or by intramuscularinjection. In certain embodiments, depot preparations are prepared usingsuitable polymeric or hydrophobic materials (for example an emulsion inan acceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

In certain embodiments, a pharmaceutical composition of the presentinvention comprises a delivery system. Examples of delivery systemsinclude, but are not limited to, liposomes and emulsions. Certaindelivery systems are useful for preparing certain pharmaceuticalcompositions including those comprising hydrophobic compounds. Incertain embodiments, certain organic solvents such as dimethylsulfoxideare used.

In certain embodiments, a pharmaceutical composition of the presentinvention comprises one or more tissue-specific delivery moleculesdesigned to deliver the one or more pharmaceutical agents of the presentinvention to specific tissues or cell types. For example, in certainembodiments, pharmaceutical compositions include liposomes coated with atissue-specific antibody.

In certain embodiments, a pharmaceutical composition of the presentinvention comprises a co-solvent system. Certain of such co-solventsystems comprise, for example, benzyl alcohol, a nonpolar surfactant, awater-miscible organic polymer, and an aqueous phase. In certainembodiments, such co-solvent systems are used for hydrophobic compounds.A non-limiting example of such a co-solvent system is the VPD co-solventsystem, which is a solution of absolute ethanol comprising 3% w/v benzylalcohol, 8% w/v of the nonpolar surfactant Polysorbate 80™ and 65% w/vpolyethylene glycol 300. The proportions of such co-solvent systems maybe varied considerably without significantly altering their solubilityand toxicity characteristics. Furthermore, the identity of co-solventcomponents may be varied: for example, other surfactants may be usedinstead of Polysorbate 80™; the fraction size of polyethylene glycol maybe varied; other biocompatible polymers may replace polyethylene glycol,e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides maysubstitute for dextrose.

In certain embodiments, a pharmaceutical composition of the presentinvention comprises a sustained-release system. A non-limiting exampleof such a sustained-release system is a semi-permeable matrix of solidhydrophobic polymers. In certain embodiments, sustained-release systemsmay, depending on their chemical nature, release pharmaceutical agentsover a period of hours, days, weeks or months.

In certain embodiments, a pharmaceutical composition of the presentinvention is prepared for oral administration. In certain of suchembodiments, a pharmaceutical composition is formulated by combining oneor more compounds comprising a modified oligonucleotide with one or morepharmaceutically acceptable carriers. Certain of such carriers enablepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions and the like, fororal ingestion by a subject. In certain embodiments, pharmaceuticalcompositions for oral use are obtained by mixing oligonucleotide and oneor more solid excipient. Suitable excipients include, but are notlimited to, fillers, such as sugars, including lactose, sucrose,mannitol, or sorbitol; cellulose preparations such as, for example,maize starch, wheat starch, rice starch, potato starch, gelatin, gumtragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). In certainembodiments, such a mixture is optionally ground and auxiliaries areoptionally added. In certain embodiments, pharmaceutical compositionsare formed to obtain tablets or dragee cores. In certain embodiments,disintegrating agents (e.g., cross-linked polyvinyl pyrrolidone, agar,or alginic acid or a salt thereof, such as sodium alginate) are added.

In certain embodiments, dragee cores are provided with coatings. Incertain such embodiments, concentrated sugar solutions may be used,which may optionally contain gum arabic, talc, polyvinyl pyrrolidone,carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquersolutions, and suitable organic solvents or solvent mixtures. Dyestuffsor pigments may be added to tablets or dragee coatings.

In certain embodiments, pharmaceutical compositions for oraladministration are push-fit capsules made of gelatin. Certain of suchpush-fit capsules comprise one or more pharmaceutical agents of thepresent invention in admixture with one or more filler such as lactose,binders such as starches, and/or lubricants such as talc or magnesiumstearate and, optionally, stabilizers. In certain embodiments,pharmaceutical compositions for oral administration are soft, sealedcapsules made of gelatin and a plasticizer, such as glycerol orsorbitol. In certain soft capsules, one or more pharmaceutical agents ofthe present invention are be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added.

In certain embodiments, pharmaceutical compositions are prepared forbuccal administration. Certain of such pharmaceutical compositions aretablets or lozenges formulated in conventional manner.

In certain embodiments, a pharmaceutical composition is prepared foradministration by injection (e.g., intravenous, subcutaneous,intramuscular, etc.). In certain of such embodiments, a pharmaceuticalcomposition comprises a carrier and is formulated in aqueous solution,such as water or physiologically compatible buffers such as Hanks'ssolution, Ringer's solution, or physiological saline buffer. In certainembodiments, other ingredients are included (e.g., ingredients that aidin solubility or serve as preservatives). In certain embodiments,injectable suspensions are prepared using appropriate liquid carriers,suspending agents and the like. Certain pharmaceutical compositions forinjection are presented in unit dosage form, e.g., in ampoules or inmulti-dose containers. Certain pharmaceutical compositions for injectionare suspensions, solutions or emulsions in oily or aqueous vehicles, andmay contain formulatory agents such as suspending, stabilizing and/ordispersing agents. Certain solvents suitable for use in pharmaceuticalcompositions for injection include, but are not limited to, lipophilicsolvents and fatty oils, such as sesame oil, synthetic fatty acidesters, such as ethyl oleate or triglycerides, and liposomes. Aqueousinjection suspensions may contain substances that increase the viscosityof the suspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, such suspensions may also contain suitablestabilizers or agents that increase the solubility of the pharmaceuticalagents to allow for the preparation of highly concentrated solutions.

In certain embodiments, a pharmaceutical composition is prepared fortransmucosal administration. In certain of such embodiments penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

In certain embodiments, a pharmaceutical composition is prepared foradministration by inhalation. Certain of such pharmaceuticalcompositions for inhalation are prepared in the form of an aerosol sprayin a pressurized pack or a nebulizer. Certain of such pharmaceuticalcompositions comprise a propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In certain embodiments using a pressurized aerosol,the dosage unit may be determined with a valve that delivers a meteredamount. In certain embodiments, capsules and cartridges for use in aninhaler or insufflator may be formulated. Certain of such formulationscomprise a powder mixture of a pharmaceutical agent of the invention anda suitable powder base such as lactose or starch.

In certain embodiments, a pharmaceutical composition is prepared forrectal administration, such as a suppositories or retention enema.Certain of such pharmaceutical compositions comprise known ingredients,such as cocoa butter and/or other glycerides.

In certain embodiments, a pharmaceutical composition is prepared fortopical administration. Certain of such pharmaceutical compositionscomprise bland moisturizing bases, such as ointments or creams.Exemplary suitable ointment bases include, but are not limited to,petrolatum, petrolatum plus volatile silicones, and lanolin and water inoil emulsions. Exemplary suitable cream bases include, but are notlimited to, cold cream and hydrophilic ointment.

In certain embodiments, a pharmaceutical composition of the presentinvention comprises a modified oligonucleotide in a therapeuticallyeffective amount. In certain embodiments, the therapeutically effectiveamount is sufficient to prevent, alleviate or ameliorate symptoms of adisease or to prolong the survival of the subject being treated.Determination of a therapeutically effective amount is well within thecapability of those skilled in the art.

In certain embodiments, one or more modified oligonucleotides of thepresent invention is formulated as a prodrug. In certain embodiments,upon in vivo administration, a prodrug is chemically converted to thebiologically, pharmaceutically or therapeutically more active form of amodified oligonucleotide. In certain embodiments, prodrugs are usefulbecause they are easier to administer than the corresponding activeform. For example, in certain instances, a prodrug may be morebioavailable (e.g., through oral administration) than is thecorresponding active form. In certain instances, a prodrug may haveimproved solubility compared to the corresponding active form. Incertain embodiments, prodrugs are less water soluble than thecorresponding active form. In certain instances, such prodrugs possesssuperior transmittal across cell membranes, where water solubility isdetrimental to mobility. In certain embodiments, a prodrug is an ester.In certain such embodiments, the ester is metabolically hydrolyzed tocarboxylic acid upon administration. In certain instances the carboxylicacid containing compound is the corresponding active form. In certainembodiments, a prodrug comprises a short peptide (polyaminoacid) boundto an acid group. In certain of such embodiments, the peptide is cleavedupon administration to form the corresponding active form.

In certain embodiments, a prodrug is produced by modifying apharmaceutically active compound such that the active compound will beregenerated upon in vivo administration. The prodrug can be designed toalter the metabolic stability or the transport characteristics of adrug, to mask side effects or toxicity, to improve the flavor of a drugor to alter other characteristics or properties of a drug. By virtue ofknowledge of pharmacodynamic processes and drug metabolism in vivo,those of skill in this art, once a pharmaceutically active compound isknown, can design prodrugs of the compound (see, e.g., Nogrady (1985)Medicinal Chemistry A Biochemical Approach, Oxford University Press, NewYork, pages 388-392).

Certain Experimental Models

In certain embodiments, the present invention provides methods of usingand/or testing modified oligonucleotides of the present invention in anexperimental model. In certain embodiments, experimental models areemployed to evaluate the effectiveness of modified oligonucleotides ofthe invention for the treatment of liver cancer, including HCC. Thosehaving skill in the art are able to select and modify the protocols forsuch experimental models to evaluate a pharmaceutical agent of theinvention.

Generally, modified oligonucleotides are first tested in cultured cells.Suitable cell types include those that are related to the cell type towhich delivery of a modified oligonucleotide is desired in vivo. Forexample, suitable cell types for the study of modified oligonucleotidesfor the treatment of liver cancer include cell types derived from livercancer, such as HepG2, Hep3B, SK-Hep1, 7721, SNU-398, SNU423, SNU449,Huh7, HCCLM3 and MHT cells.

In certain embodiments, the extent to which a modified oligonucleotideinterferes with the activity of a miRNA is assessed in cultured cells.In certain embodiments, inhibition of miRNA activity may be assessed bymeasuring the levels of the miRNA. Alternatively, the level of apredicted or validated miRNA target may be measured. An inhibition ofmiRNA activity may result in the increase in the mRNA and/or protein ofa miRNA target. Further, in certain embodiments, certain phenotypicoutcomes may be measured. For example, suitable phenotypic outcomesinclude inhibition of cell proliferation, the induction of cell death,and/or the induction of apoptosis. Other suitable phenotypic outcomesinclude the arrest of cells at any point of the cell cycle, such as theG1/S transition, S phase, the G2/M transition, mitotic division, orcytokinesis.

Following the in vitro identification of a modified oligonucleotide thateffectively inhibits the activity of a miRNA, modified oligonucleotidesare further tested in in vivo experimental models. Suitable experimentalmodels for the testing of chemotherapeutic agents, including modifiedoligonucleotides complementary to a miRNA described herein, include: asubcutaneous xenograft mouse model, an orthotopic liver xenograft mousemodel, an SV40 t/T transgenic mouse model, a TGF-α/c-myc transgenicmouse model and a chemically induced carcinogenic (diethylnitrosamine)mouse model.

A suitable in vivo experimental model for the testing of modifiedoligonucleotides of the present invention includes the subcutaneousxenograft mouse model. In this model, cells are removed from culture andinjected subcutaneously into mice. Suitable cells include, for example,Hep3B cells. Suitable mice include, for example, BALB/c nude mice. Asuitable injection site is, for example, the flank of the mouse.Modified oligonucleotide, dissolved in saline, is administered to themice at a frequency of 2 to 3 times per week. Modified oligonucleotideis administered prior to implantation, simultaneously with implantation,or after implantation. Suitable administration route includeintraperitoneal administration and intratumoral administration. Modifiedoligonucleotide doses range from 5 to 50 mg/kg. The animals are treatedfor 3 to 4 weeks, after which tumor size, tumor number, and liver weightare measured. Measurements may be made with digital calipers.Saline-treated animals are used as a control group. A chemotherapeuticagent, such as, for example, 5-flurouracil, may be used as a positivecontrol for the inhibition of tumor size or number. Various endpointsare assessed, including tumor size, tumor number, and liver weight.Modified oligonucleotide-treated mice are compared to the same endpointsin control-treated mice. Statistical analyses are employed to identifysignificant differences in any of the endpoints. The subcutaneousxenograft model is an art-accepted model for the in vivo evaluation ofchemotherapeutic agents, including modified oligonucleotides. See, forexample, Koller et al., Cancer Res., 2006, 66, 2059-2066, and Cheng etal., Cancer Res., 2007, 67, 309-317.

A suitable in vivo experimental model for the testing of modifiedoligonucleotides of the present invention is the HCCLM3 orthotopic liverxenograft model. In this model, approximately 1 million HCCLM3 cells (ahighly metastatic human HCC cell line) are subcutaneously injected intothe flanks of BALB/c nude mice. Once tumors are an appropriate size(e.g. 1 mm³), tumor fragments are removed and intrahepatically implantedinto other BALB/c nude mice. At this point, modified oligonucleotide,dissolved in saline, is administered to the mice at a frequency of 2 to3 times per week. Alternatively, administration of modifiedoligonucleotide begins several days (e.g. 10 days) followingimplantation. Suitable administration route include intraperitonealadministration and intratumoral administration. Modified oligonucleotidedoses range from 5 to 50 mg/kg. The animals are treated for 3 to 4 weeksfor a short term study, after which tumor size, tumor number, and liverweight are measured. Alternatively, the animals are treated for 8 to 30weeks for a long term study, after which various endpoints are assessed,including tumor size, tumor number, liver weight, number of metastasesand survival will be measured. Metastasis is measured in tissues such aslung tissue. Measurements of tumor size and weight may be made withdigital calipers. Saline-treated animals are used as a control group. Achemotherapeutic agent, such as, for example, 5-flurouracil, may be usedas a positive control for the inhibition of tumor size or number.Endpoints observed in modified oligonucleotide-treated mice are comparedto the same endpoints in control-treated mice. Statistical analyses areemployed to identify significant differences in any of the endpoints.The orthotopic xenograft model is an art-accepted model for the in vivoevaluation of chemotherapeutic agents, including modifiedoligonucleotides. See, for example, Li et al., Clin. Cancer Res., 2006,12, 7140-7148. As an alternative to HCCLM3 cells, HepG2 cells may beused to establish the orthotopic model.

An additional suitable in vivo experimental model is the SV40 t/Ttransgenic mouse model. Transgenic mice have been engineered to expressthe SV40 large T antigen (SV40 t/T mice) under the control of theliver-specific C-reactive protein promoter (Ruther et al., Oncogene,1993, 8, 87-93). The expression of SV40 large T antigen can betransiently induced by injection of bacterial lipopolysaccacharide, andresults in the development of hepatocellular carcinoma. At this point,modified oligonucleotide, dissolved in saline, is administered to themice at a frequency of 2 to 3 times per week. Modified oligonucleotidedoses range from 5 to 50 mg/kg. Suitable administration route includeintraperitoneal administration and intratumoral administration. Theanimals are treated for 3 to 4 weeks for a short term study, after whichtumor size, tumor number, and liver weight are measured. Alternatively,the animals are treated for 8 to 30 weeks for a long term study, afterwhich various endpoints are measured, including tumor size, tumornumber, liver weight, number of metastases, and survival. Metastasis ismeasured in tissues such as lung tissue. Measurements of tumor size andweight may be made with digital calipers. Saline-treated animals areused as a control group. A chemotherapeutic agent, such as, for example,5-flurouracil, may be used as a positive control for the inhibition oftumor size or number. Endpoints observed in modifiedoligonucleotide-treated mice are compared to the same endpoints incontrol-treated mice. Statistical analyses are employed to identifysignificant differences in any of the endpoints.

A suitable in vivo experimental model is a chemically-inducedcarcinogenic mouse model. In this model, liver cancer is induced byadministration of the carcinogen diethylnitrosamine (DEN). Mice areinjected intraperitoneally with 5 or 25 mg/kg DEN. Modifiedoligonucleotide, dissolved in saline, is administered to the mice at afrequency of 2 to 3 times per week. Modified oligonucleotide doses rangefrom 5 to 50 mg/kg. Suitable administration route includeintraperitoneal administration and intratumoral administration. Theanimals are treated for 4 to 8 weeks for a short term study, after whichtumor size, tumor number, and liver weight are measured. Alternatively,the animals are treated for 8 to 30 weeks for a long term study, afterwhich tumor size, tumor number, liver weight, number of metastases andsurvival will be measured. Metastasis is measured in tissues such aslung tissue. Measurements of tumor size and weight may be made withdigital calipers. Saline-treated animals are used as a control group. Achemotherapeutic agent, such as, for example, 5-flurouracil, may be usedas a positive control for the inhibition of tumor size or number.Endpoints observed in modified oligonucleotide-treated mice are comparedto the same endpoints in control-treated mice. Statistical analyses areemployed to identify significant differences in any of the endpoints.The DEN-induced HCC model has been used for the study of HCC. See, forexample, Maeda et al., Cell, 2005, 121, 977-990.

Dioxins

Dioxins are a family of environmental pollutants such as2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), that are known to havemultiple hazardous effects. TCDD is known to be a most potentcarcinogen, and also to induce other adverse biological responses.Dioxin induced effects include, but are not limited to, skin diseases,birth defects, miscarriages, developmental defects, teratogenesis,immunotoxicity and cancer. Dioxins are produced in small concentrationswhen organic material is burned in the presence of chlorine. Thisprocedure occurs often in a variety of industrial processes such as inthe bleaching of paper, but dioxins can also be produced from naturalsources such as volcanoes and forest fires. Dioxins enter the generalpopulation primarily from ingestion of food (herbicides), due to theirlipophilic properties, but also by inhalation. The general treatmentafter dioxin exposure is dietary fat to remove it from the body since itis very lypophilic. Additional approaches for lowering dioxin includedietary intake of mineral oil (Moser and McLachlan, 1999), activatedcharcoal (Araki, 1974), rice bran oil (Ilda, 1995), or the fatsubstitute Olestra (Geusau et al., 1999, 2002), however theeffectiveness of these treatments is minimal.

The mechanism of dioxins' carcinogenic effect is not yet fullyunderstood, however it is known to be an Aryl hydrocarbon receptor (AhR)ligand, and most, if not all of its effects, are thought to be mediatedthrough the activation of AhR.

AhR belongs to a family of ligand activated transcription factors basichelix-loop-helix/Per-Arnt-Sim (bHLH/PAS) that mediates transcriptionalactivation of sets of enzymes that function in the metabolism ofxenobiotics. Upon ligand binding the AhR translocates to the nucleus andassociates with its partner protein Arnt to form a heterodimer. Theheterodimer binds to an enhancer site on the DNA designated xenobioticresponsive element (XRE) and is responsible to regulate a variety oftranscription activation of enzymes involved in xenobiotic metabolismand other functions. One of the genes that are transcriptionallyregulated by AhR is an AhR repressor (AhRR) that can also form aheterodimer with Arnt and bind to XRE, however this formstranscriptional repression. Since AhRR transcription is regulated byAhR, AhR and AhRR form a regulatory feedback loop.

As a result of AhR activation an “AhR gene battery” of Phase I and PhaseII metabolizing enzymes consisting of CYP1A1, CYP1A2, CYP1B1, NQO1,ALHD3A1, UGT1A2 and GSTA1 is up-regulated. This response presumablyevolved to be able to detect a wide range of chemicals, indicated by thewide range of substrates AhR is able to bind and facilitate theirbiotransformation and elimination as detoxification process.

However, AhR activation also elicits toxic responses. Toxicity resultsfrom two different pathways of AhR signaling. The first is when theinduction of metabolizing enzymes results in the production of toxicintermediate metabolites. The second path to toxicity is the result ofaberrant changes in global gene transcription beyond those observed inthe “AhR gene battery.” These global changes in gene expression lead toadverse changes in cellular processes and function.

Many studies conducted in order to elucidate the mechanism andunderstand the toxicity and carcinogenicity of TCDD via AhR activationresulted with paradoxical outcomes. Repeatedly inconsistent results arereported, showing both apoptotic and anti-apoptotic effects of TCDDactivated AhR cellular responses, usually explained by differences inthe treatment regiment and models tested.

Although the induction of the AhR by dioxins is well characterized, thefunction and mechanism of some of its toxicities are still unknown andthe paradoxical and contradicting results appearing in many articlesindicate the necessity for further study of TCDD mechanism ofcarcinogenicity.

To date there has been no definitive description of any miR whoseexpression is directly regulated by dioxins, or of the functionalconsequences of such regulation; Moffat et al. (Toxicol Sci. 2007October; 99(2):470-87) showed only very moderate changes in miRs inresponse to TCDD in rodent models and concluded that microRNAs do notplay a role in dioxin toxicity.

As demonstrated herein, hsa-miR-191, which is up-regulated in HCC, isalso up-regulated after TCDD activation of the AhR transcription factor,together with miR-181a, and hsa-miR-181b, and to a lesser degreehsa-miR-181a*. Thus, the AhR transcription factor is responsible for theregulation of the expression of miRs having an AhR TFBS motif at theirpromoters. The involvement of miRs in the mechanism of TCDD activity canexplain the down regulation of several genes as seen on expressionarrays, apart from transcriptional activation through AhR.

Certain Quantitation Assays

The effects of antisense inhibition of a miRNA following theadministration of modified oligonucleotides may be assessed by a varietyof methods known in the art. In certain embodiments, these methods arebe used to quantitate miRNA levels in cells or tissues in vitro or invivo. In certain embodiments, changes in miRNA levels are measured bymicroarray analysis. In certain embodiments, changes in miRNA levels aremeasured by one of several commercially available PCR assays, such asthe TaqMan® MicroRNA Assay (Applied Biosystems). In certain embodiments,antisense inhibition of a miRNA is assessed by measuring the mRNA and/orprotein level of a target of a miRNA. Antisense inhibition of a miRNAgenerally results in the increase in the level of mRNA and/or protein ofa target of the miRNA.

The following examples are presented in order to more fully illustratesome embodiments of the invention. They should, in no way be construed,however, as limiting the broad scope of the invention.

EXAMPLES Example 1: Expression Profiling of miRNAs in Tissue Samples

To identify miRNAs that are dysregulated in association with cancer,miRNA expression profiles were analyzed in liver samples from subjectswith hepatocellular carcinoma (HCC), and were compared to expressionprofiles in normal liver. Samples analyzed included: 37 liver samplesfrom human HCC subjects; 39 liver samples of normal liver adjacent toHCC; and 2 liver samples from normal human liver. Of the 39 samples ofnormal liver adjacent to HCC, 36 were from the human HCC subjects.

Liver samples were also collected from transgenic mice which express theSV40 t/T antigen under the control of the C-reactive protein promoter.This promoter results in hepatocyte-specific expression of the oncogenicSV40 t/T antigen, which eventually leads to the development of livertumors that are histologically characterized as hepatocellularcarcinoma. Samples analyzed included: 12 samples from normal mouseliver; 18 HCC samples from SV40 transgenic mice.

Also analyzed were HCC-related cell lines, including HepG2, Hep3B,SK-Hep1, 7721, SNU-398, SNU423, SNU449, Huh7 and MHT. MHT cells areisolated from the livers of SV40 t/T antigen transgenic mice. Monkeyhepatocytes were also analyzed.

RNA was extracted from the samples using the miRvana miRNA isolation kit(Ambion) according to the manufacturer's instructions and hybridized toa microRNA array. Custom microarrays were produced by printing DNAoligonucleotide probes representing about 700 miRNAs, including miRNAsfrom the Sanger database, version 9 and additional Rosetta genomicsvalidated and predicted miRs. Each probe, printed in triplicate, carriesup to 22-nt linker at the 3′ end of the miRNA's complement sequence inaddition to an amine group used to couple the probes to coated glassslides. 20 μM of each probe were dissolved in 2×SSC+0.0035% SDS andspotted in triplicate on Schott Nexterion® Slide E coated microarrayslides using a Genomic Solutions® BioRobotics MicroGrid II according theMicroGrid manufacturer's directions. 64 negative control probes weredesigned using the sense sequences of different miRNAs. Two groups ofpositive control probes were designed to hybridize to miRdicator™ array(1) synthetic spikes small RNA were added to the RNA before labeling toverify the labeling efficiency and (2) probes for abundant small RNA(e.g. small nuclear RNAs (U43, U49, U24, Z30, U6, U48, U44), 5.8s and 5sribosomal RNA) are spotted on the array to verify RNA quality. Theslides were blocked in a solution containing 50 mM ethanolamine, 1M Tris(pH 9.0) and 0.1% SDS for 20 min at 50° C., then thoroughly rinsed withwater and spun dry.

Five μg of total RNA was labeled by ligation of a RNA-linkerp-rCrU-Cy-dye (Thomson et al., 2004, Nat Methods 1, 47-53) (Dharmacon)to the 3′-end with Cy3 or Cy5. The labeling reaction contained totalRNA, spikes (0.1-20 fmoles), 300 ng RNA-linker-dye, 15% DMSO, lx ligasebuffer and 20 units of T4 RNA ligase (NEB) and proceeded at 4° C. for 1hr followed by 1 hr at 37° C. The labeled RNA was mixed with 3×hybridization buffer (Ambion), heated to 95° C. for 3 min and than addedon top of the miRdicator™ array. Slides were hybridize 12-16 hr,followed by two washes with 1×SSC and 0.2% SDS and a final wash with0.1×SSC.

Arrays were scanned using an Agilent Microarray Scanner Bundle G2565BA(resolution of 10 μm at 100% power). Array images were analyzed usingSpotReader software (Niles Scientific).

Raw data of miRNA signals were normalized and a T-test was used toidentify statistically significant differentially expressed miRNAs.

94 miRNAs were selected as candidate miRNAs for further study. ThesemiRNAs were selected based on one or more of the following criteria:differential expression in human liver tumor samples relative to normalhuman liver samples; differential expression in mouse HCC samplesrelative to normal mouse liver samples; or high expression in humanliver tissue. FIG. 1 illustrates 8 of the miRNAs that exhibited elevatedexpression in liver tumor samples.

Example 2: miRNA Expression Profiling of Cancer Cell Lines

The miRNA expression profiles of miRNAs in various cancer cell lineswere compared to miRNA expression profiles of human liver cancersamples. It was observed that many of the miRNAs highly expressed inhuman liver cancer samples were also highly expressed in human cancercell lines. These miRNAs included, for example, miR-21, and miR-191.Accordingly, the human liver cancer cell lines are useful for theidentification and study of modified oligonucleotides that arecandidates for the treatment of liver cancer.

Example 3: Anti-Proliferative Effects of Modified Oligonucleotides

To determine the involvement of the candidate miRNAs in cellproliferation, modified oligonucleotides were used to inhibit theactivity of the candidate miRNAs.

The ability of the cells to proliferate was measured using the MTS CellProliferation Assay (CellTiter 96® AQueous One Solution CellProliferation Assay Promega Corporation Madison, Wis.). The MTS assay isa colorimetric assay that measures the reduction of a tetrazoliumcomponent (MTS reagent) into an insoluble formazan product by themitochondria of viable cells. After incubation of the cells with the MTSreagent for approximately 2 to 4 hours, the samples are read using anELISA plate reader at a wavelength of 490 nM. The amount of colorproduced is directly proportional to the number of cells.

Modified oligonucleotides complementary to the selected miRNAs weredesigned and synthesized. Each nucleoside of each modifiedoligonucleotide has a 2′-O-methoxyethyl sugar, each internucleosidelinkage is a phosphorothioate internucleoside linkage, and all cytosinesare 5-methylcytosines. Additional modified oligonucleotides testedincluded modified oligonucleotides having a 2′-O-methoxyethyl sugar ateach nucleoside, and phosphodiester internucleoside linkages.

The modified oligonucleotides were tested for their anti-proliferativeeffects in Hep3B cells and SNU423 cells. Cells were treated with 20, 40,70, 150, or 300 nM of modified oligonucleotide, in triplicate samples,for a period of 4 hours, after which the media was replaced with normalgrowth media. Oligofectamine was used as the transfection reagent.Untreated cells served as controls, as well as transfection with amodified oligonucleotide with 6 mismatches to hsa-mir-122. As a controlfor inhibition of proliferation, cells were treated with a modifiedoligonucleotide known to inhibit cell proliferation. The proliferationassay was performed 48 to 72 hours following addition of the modifiedoligonucleotides.

The number of cells in modified oligonucleotide-treated samples wascompared to the number of cells in untreated control samples. In thisway, the proliferation of cells was measured. The comparison revealedthat antisense inhibition of miR-21, miR-125a-5p, miR-191, miR-210,miR-222, miR-378, miR-423-3p, and miR-638 resulted in inhibition of cellproliferation (FIG. 2). Thus, modified oligonucleotides complementary toa miR selected from miR-21, miR-125a-5p, miR-191, miR-210, miR-222,miR-378, miR-423-3p, and miR-638 exhibited anti-proliferative effects inHCC cell lines. As shown in FIG. 1, the expression of each of these 8miRNAs is elevated in liver tumor samples, relative to normal livertissue samples. Accordingly, such modified oligonucleotides aretherapeutic agents for the treatment of HCC. Examples of such modifiedoligonucleotides are illustrated in Table 1.

Example 4: Apoptotic Activity of Modified Oligonucleotides

To determine the involvement of the candidate miRNAs in cell survival,modified oligonucleotides were used to inhibit the activity of themiRNAs, and caspase activity was used as an indicator of apoptosis.

Apoptosis was evaluated by measuring the activity of caspase 3 andcaspase 7. A fluorogenic substrate was added to the wells of cells. Whenthis substrate is cleaved by activated caspases 3 and 7, a fluorescentsignal is generated. This signal can be quantitated in a fluorescenceplate reader and used to determine the extent of capsase activation.

The modified oligonucleotides shown in Table 1 were tested for theireffects on caspase 3 and caspase 7 activity in Hep3B cells. Cells weretreated with 50, 100, 150, or 200 nM of modified oligonucleotide, intriplicate samples, for a period of 24 hours. Oligofectamine was used asthe transfection reagent. Untreated cells served as controls as well astransfection with a modified oligonucleotide having 6 mismatches tohas-miR-122.

The caspase 3/7 activity in oligonucleotide-treated samples was comparedto the caspase 3/7 activity in untreated control samples. In this way,the induction of apoptosis was measured. The comparison revealed thatantisense inhibition of miR-21, miR-125a-5p, miR-191, miR-210, miR-378,miR-423-3p, and miR-638 resulted in increased caspase 3/7 activity (FIG.3). Thus, modified oligonucleotides complementary to a miR selected frommiR-21, miR-125a-5p, miR-191, miR-210, miR-378, miR-423-3p, and miR-638induced apoptosis in Hep3B cells. Accordingly, such modifiedoligonucleotides are therapeutic agents for the treatment of HCC.

Example 5: Anti-Tumor Effects of Modified Oligonucleotides In Vivo

To determine the effects of modified oligonucleotides targeted to miRNAson tumor growth, modified oligonucleotides were evaluated in a mousemodel of hepatocellular carcinoma. In this mouse model, HCC-derivedcells injected into nude mice form tumors, and modified oligonucleotidesare tested for their ability to slow and/or inhibit tumor growth.

To induce tumor formation, a solution containing approximately 5×10⁶HepG2 cells suspended in Matrigel was injected subcutaneously into nudemice.

The modified oligonucleotides tested in this model included:MOE-modified anti-miR-21, a modified oligonucleotide targeted to miR-21having a 2′-MOE modification at each sugar, phosphorothioateinternucleoside linkages throughout, where each cytosine is a 5-methylcytosine; and MOE-modified anti-miR-210 having a 2′-MOE modification ateach sugar, phosphorothioate internucleoside linkages throughout, whereeach cytosine is a 5-methyl cytosine. Phosphate-buffered saline (PBS)was used as a control treatment.

Treatment groups were as follows: (1) control; (2) 50 mg/kg MOE-modifiedanti-miR21; (3) 50 mg/kg MOE-modified anti-miR-210. Each treatment groupcontained 10 mice. Mice received intraperitoneal injections of controlor modified oligonucleotide beginning on day 4 following tumor inductionand continuing every other day for a total of 12 injections (i.e. days4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26). Tumor size wasmonitored with calipers on days 12, 15, 18, 22, 25, and 28 followingtumor induction. Tumor volume was calculated as (L*W²)/2, where L=length(mm) and W=width (mm). Mean tumor volumes for modifiedoligonucleotide-treated groups were compared to mean tumor volumes forcontrol-treated groups; fold changes in mean tumor volume are shown inTable 7 and FIG. 4. P-values were calculated by t-test.

TABLE 7 Days post Fold change in tumor mean tumor Treatment inductionvolume p-value MOE-modified anti-miR-21 12 2 0.0466 15 1.9 0.0109 18 1.90.0067 22 1.6 0.0251 25 1.2 0.2973 28 1.2 0.2785 MOE-modifiedanti-miR-210 12 3.9 0.0004 15 2 0.0006 18 1.6 0.0113 22 1.3 0.1531 251.1 0.4919 28 1.1 0.3646

As shown in Table 7, treatment with 50 mg/kg MOE-modified anti-miR-21resulted in statistically significant smaller tumor size at days 12, 15,18, and 22 following tumor induction, relative to tumor size incontrol-treated mice. Reductions in tumor size were also observed atdays 25 and 28 following tumor induction. Similarly, treatment with 50mg/kg MOE-modified anti-miR-210 resulted in statistically significantsmaller tumor size at days 12, 15, 18, and 22 following tumor induction,relative to tumor size in control-treated mice. Reductions in tumor sizewere also observed at days 25 and 28 following tumor induction.Accordingly, modified oligonucleotides complementary to miR-21 andmiR-210 are therapeutic agents for the treatment of HCC.

Example 6: Induction of miR Expression by Activation of the AhR TF byTCDD

HCC cells treated with TCDD were studied for miR expression on amicroarray (microarray analysis was performed as described in Example1). As demonstrated in FIG. 5, expression of each of hsa-miR-191,hsa-miR-181a, hsa-miR-181b and hsa-miR-181a* was shown to be elevatedmore than twofold in TCDD treated cells after 48 hours, compared tountreated cells.

Example 7: Dual-Luciferase Reporter Assay for miR-191

A dual-luciferase reporter assay was prepared to evaluate miR-191activity. Custom-made 42-nucleotide long complementary oligonucleotides(IDT) were designed to be inserted into the 3′ UTR of renilla luciferasein a psiCHECK-2 vector (Promega); these oligonucleotides included thereverse complement sequence to selected miRs. Complementaryoligonucleotides were annealed, creating NotI and XhoI sticky ends.Sequences included the relevant reverse complement miR sequences and onenegative control. These inserts were designed to create miR bindingsites, and each insert was cloned in the 3′UTR of renilla luciferase ina psiCHECK-2 vector. Clones were verified in three stages: (1) colonyPCR, (2) restriction with HindIII utilizing the site added with theinsert, and (3) sequencing. SNU423 cells were transfected in triplicateswith either one of the vectors or co-transfected with a vector and anASO using Lipofectamine2000 reagent (Invitrogen, Cat#11668027)Luminescence was assayed 24 and 48 hours later using the Dual-LuciferaseReporter Assay System (Promega, Cat#E1961) according to manufacturer'sinstructions, on “The Reporter” microplate luminometer (Turner designs).Results were normalized to the constitutively expressed fireflyluciferase from the same vector, and presented as the ratio between thevarious treatments and cells transfected with a non-modified vector.

As indicated in FIG. 6, endogenous hsa-miR-191 (bar a) indeeddownregulates the reporter expression, and this effect is almostcompletely abolished by co-transfection of the reporter vector togetherwith the antisense oligonucleotide inhibiting hsa-miR-191 (bar b). Thebar-chart further shows the specificity of the response, since anothercontrol ASO could not abolish the miR regulation of the reporter (barc), and the endogenous miR did not change the expression of the reporteron a control plasmid having an altered 3′ UTR but with a non-relevantsequence, with (bar d) or without an ASO (bar e).

Example 8: AhR/Arnt and Regulation of Hsa-miR-191

Transcription factor binding site (TFBS) motifs were searched for atlocations +/−1000 bp from the Transcription Start Site of hsa-miR-191.The AhR/Arnt TFBS was predicted at the following location:

#hg18.tfbs hg18.tfbsCo hg18.tfbsCon hg18.tfbsConsSites.name hg18.tfbsChg18.tfbsC hg18.tfbsC hg18.tfbsConsFactors.id Chr3 49034918 49034937V$AHRARNT_02 + 2.42 AhR, Arnt, P35869, P27540,

A ChIP (Chromatin Immuno Precipitation) assay was conducted to validatethe predicted TFBS and the involvement of this TF in the transcriptionalregulation of hsa-miR-191.

The ChIP assay was performed as follows:

HepG2 cells were treated with TCDD at 10 nM concentration. Cells werethen fixed when freshly-prepared 11% Formaldehyde Solution was added tothe existing media.

Fixation was stopped by adding Glycine Solution. Cells are then scrapedoff from the culture surface, washed in chilled PBS-Igepal and treatedwith 1 mM PMSF. Cells are finally centrifuged and pellet is snap-frozen.

The immunoprecipitation is done at Genepathway and the binding ofChromatin to the precipitated TF was quantified by qPCR.

Data values were generated using a standard curve of genomic DNA withknown copy numbers. Positive controls are genomic regions containingknown binding sites for the factor under investigation, and the negativecontrols are genomic regions not bound by the factor underinvestigation. Analysis was done in triplicates.

Input DNA values (unprecipitated genomic DNA) were used to calculate thePrimer Efficiency Ratio for every primer pair relative to the primerpair used in the standard curve. The data was presented as the BindingEvents Per 1000 Cells for each genomic region tested. These values,which are calculated from the average of the triplicate qPCR values foreach test, take into account the amount of chromatin that wasimmunoprecipitated plus the proportion tested by qPCR, and arenormalized for primer efficiency. Also the standard deviations for eachtest are calculated, which have been normalized in the same way as thetest values.

Genpathway has demonstrated that changes in factor binding as low as1.3× can be reproducibly determined in a variety of biological systems.Therefore, genomic regions showing fold differences of 1.5 or greaterare considered significant.

Since TCDD is a known ligand of AhR and activates this TF to induce theexpression of CYP1 proteins, TCDD treatment was included as an activatorfor the TF, and CYP1A1 was chosen as a control gene in the ChIP assay.CYP1A1 has two TFBSs for the AhR/Arnt TF, both which were tested.

As seen in FIG. 7, which summarizes the results of the ChIP assay usinga specific antibody for the AhR TF, AhR was found to bind to thepromoter of the hsa-miR-191 transcript Similar results were achievedwhen a ChIP assay was conducted with an Ab against Arnt, which indicatesthe activity of the heterodimer AhR/Arnt.

The foregoing description of the specific embodiments so fully revealsthe general nature of the invention that others can, by applying currentknowledge, readily modify and/or adapt for various applications suchspecific embodiments without undue experimentation and without departingfrom the generic concept, and, therefore, such adaptations andmodifications should and are intended to be comprehended within themeaning and range of equivalents of the disclosed embodiments. Althoughthe invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

What is claimed is:
 1. A method for treating liver cancer comprisingadministering to a subject in need thereof (i) a compound comprising amodified oligonucleotide consisting of 15 to 30 linked nucleosides,wherein the modified oligonucleotide has a nucleobase sequence that is100% identical over a region of 8 or more nucleobases of a sequenceselected from SEQ ID NOs: 18 and 19 and (ii) at least one additionaltherapy, wherein the at least one additional therapy is achemotherapeutic agent.
 2. The method of claim 1, wherein the livercancer is hepatocellular carcinoma.
 3. The method of claim 1, whereinthe subject is a human.
 4. The method of claim 1, wherein the compoundconsists of a modified oligonucleotide.
 5. The method of claim 1,wherein the modified oligonucleotide has a nucleobase sequencecomprising at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguousnucleobases of a nucleobase sequence selected from SEQ ID NOs: 18 and19.
 6. The method of claim 1, wherein the modified oligonucleotide has anucleobase sequence consisting of a nucleobase sequence selected fromSEQ ID NOs: 18 and
 19. 7. The method of claim 1, wherein at least oneinternucleoside linkage is a modified internucleoside linkage.
 8. Themethod of claim 1, wherein the modified internucleoside linkage is aphosphorothioate internucleoside linkage.
 9. The method of claim 1,wherein each internucleoside linkage is a modified internucleosidelinkage.
 10. The method of claim 9, wherein the modified internucleosidelinkage is a phosphorothioate internucleoside linkage.
 11. The method ofclaim 1, wherein at least one nucleoside comprises a modified sugar. 12.The method of claim 11, wherein each modified sugar is independentlyselected from a 2′-O-methoxyethyl sugar, a 2′-fluoro sugar, a2′-O-methyl sugar, or a bicyclic sugar moiety.
 13. The method of claim1, wherein each nucleoside comprises a modified sugar.
 14. The method ofclaim 13, wherein each modified sugar is independently selected from a2′-O-methoxyethyl sugar, a 2′-fluoro sugar, a 2′-O-methyl sugar, or abicyclic sugar moiety.
 15. The method of claim 1, wherein at least onenucleoside comprises a modified nucleobase.
 16. The method of claim 15,wherein the modified nucleobase is a 5-methylcytosine.
 17. The method ofclaim 1 wherein the chemotherapeutic agent is selected from5-fluorouracil, gemcitabine, doxorubicine, mitomycin c, sorafenib,etoposide, carboplatin, epirubicin, irinotecan, and oxaliplatin.
 18. Themethod of claim 1, wherein the administering results in reduction oftumor size or tumor number, or wherein the administering prevents anincrease in tumor size or tumor number.
 19. The method of claim 1wherein the administering prevents or slows metastatic progression. 20.The method of claim 1 wherein the administering extends overall survivaltime or progression-free survival time of the subject.
 21. The method ofclaim 1 wherein the subject has elevated serum alpha-fetoprotein orelevated serum des-gamma-carboxyprothrombin.
 22. The method of claim 21wherein the administering reduces serum alpha-fetoprotein or serumdes-gamma-carboxyprothrombin.
 23. The method of claim 1 wherein themodified oligonucleotide has a nucleobase sequence comprising at least17, 18, 19, 20, or 21 contiguous nucleobases of a nucleobase sequenceselected from SEQ ID NOs: 18 and
 19. 24. The method of claim 1 whereinthe nucleobase sequence of the modified oligonucleotide has nomismatches to the nucleobase sequence of SEQ ID NO: 10.